CH676247A5 - - Google Patents

Abstract

Recombinant viruses which direct the expression of peptides or proteins related to epitopes of LAV/HTLV III, the etiological agent of Lymphadenopathy Syndrome and Acquired Immune Deficiency Syndrome. These viruses can be formulated as viral vaccines or in multivalent vaccines that protect against LAV/HTLV III infection. Antigenic peptides and proteins related to epitopes of LAV/HTLV III are also described. These can be prepared using recombinant DNA techniques or by chemical synthesis and can be used as immunogens in subunit vaccines or as antigens in diagnostic immunoassays.

Description

       

  
 



  The present invention relates to a recombinant virus allowing the preparation of vaccines against acquired immunodeficiency syndrome (AIDS), as well as the vaccines obtained and, also, the various intermediates used for this obtaining and their preparation methods.



  Thus, the present invention relates to viruses which express peptides and proteins related to epitopes of the virus associated with lymphadenopathy [Lymphadenopathy Associated Virus (LAV)] / human T-cell leukemia virus [Human T-Cell Leukemia Virus ( HTLV-III)], the etiological agent of the lymphadenopathy syndrome (LAS) and of the acquired immunodeficiency syndrome (AIDS); [Acquired Immune Deficiency Syndrome (AIDS)]. The viruses of the present invention which express LAV / HTLV-III related peptides inducing an immunoprotection response may serve as immunogens in virus formulations for adenopathy syndrome (LAS) or AIDS or in multivalent vaccine formulations.

  In fact, the infectious viruses of the present invention, which can multiply in a host without causing disease, can be used in vaccine formulations based on live viruses which provide prolonged immunogenic stimulation and can give rise to strong immunity.



  The present invention also includes peptides and proteins related to LAV / HTLV III epitopes, which can be used as immunogens in vaccine formulations with subunit (s) for LAS or AIDS, or in multivalent vaccine formulations, or as antigens in immunoassays for the diagnosis of LAS or AIDS. These peptides and proteins can be produced using techniques using recombinant DNA in any vector-host system, or they can be synthesized by chemical methods.

  Therefore, the invention also includes the construction of new DNA sequences and vectors comprising plasmid DNA, and viral DNA such as viruses infecting humans, viruses infecting animals, viruses infecting insects or bacteriophages which can be used to direct the expression of peptides and proteins related to LVA / HTLV in cells of an appropriate host from which the peptides and proteins can be purified. Chemical methods for the synthesis of LAV / HTLV III related peptides and proteins are also described.



  In a specific embodiment included in the present invention, recombinant vaccinia viruses have been used to produce proteins related to the envelope or central part (core) of LAV / HTLV III. For this, envelope or gag DNA sequences encoding the envelope glycoproteins or the core structure proteins of LAV / HTLV III, respectively, were inserted into vaccinia vectors capable of directing the expression of LAV / HTLV III genes in an appropriate host. The LAV / HTLV III envelope related proteins produced by recombinant vaccinia viruses have been found to be antigenic and immunogenic and capable of eliciting humoral and cell-mediated immunity in subhuman primates.

  The LAV / HTLV III gag-like proteins produced by the recombinant vaccinia virus are immunoreactive proteins that contain the major epitopes of authentic nucleus proteins.



  Recombinant vaccinia viruses, which express proteins related to the LAV / HTLV III envelope, can be used in isolation or in concert with vaccinia recombinants which express other proteins related to LAV / HTLV III (such as structural proteins nucleus) in virus-based vaccine formulations. Alternatively, the LAV / HTLV III related proteins produced by the recombinant viruses can be purified or chemically synthesized, and used as immunogens in vaccine subunit (s) formulations. Since the LAV / HTLV III related glycoprotein (s) may be recognized as "foreign" in the animal host, a humoral and / or cellular mediated immune response will be elicited against this protein or combination of proteins.

  In an appropriately prepared vaccine formulation, this should protect the host from subsequent LAV / HTLV III infections.



  In another specific embodiment of the present invention, recombinant bacculoviruses (Autoqrapha Californica, nuclear polyhedrosis virus or ANCPV) were used to produce envelope and core related proteins of LAV / HTLV III. For this, we have inserted DNA sequences of envelope or gag, which code for proteins of envelope or nucleus structure, respectively, in vectors of the bacculovirus type which are capable of directing the expression of the genes of LAV / HTLV III in an appropriate host. The LAV / HTLV III proteins, produced by the recombinant bacculoviruses, have been found to be antigenic, based on measurement by radioimmunoprecipitation and ELISA.



  The present invention also includes the production of LAV / HTLV III antigens, which are of general importance in human medicine. This includes the use of the peptides and proteins of the present invention as reagents in immunoassays such as enzyme linked immunosorbent assay (ELISA) and radioimmunoassays which are useful as diagnostic tools for the detection of antibodies. specific for LAV / HTLV in blood samples, body fluids, tissues, etc. In addition, this reagent will be an interesting tool to elucidate the mechanism of the pathogenesis of LAV / HTLV III.



  AIDS (Acquired Immune Deficiency Syndrome) is a disease characterized by a serious immune deficiency mainly due to disorders of the cell-mediated immune response of the patient (M. Gottlieb et al., 1981, N. Engl. J. Med. 305 : 1425; J Masur et al., 1981, N. Engl. J. Med. 305: 431).

   Two clinical presentations of the disease are recognized: (a) a prodrome phase, called lymphadenopathy syndrome (LAS), characterized by chronic lymphadenopathy, leukopenia and a quantitative decrease in peripheral blood helper cells ( OKT4 cells), leading to an inversion of the normal ratio between helper and suppressor T cells (OKT4: OKT8), which moves 2 to 0.1 or less as the disease progresses; and (b) an immunodeficiency characterized by a decrease in OKT4 cells and the reversal of the normal OKT4: OKT8 ratio, absolute lymphopenia, and repetitive infections mainly due to Pneumocystis carnii; this last phase is ultimately associated with death in the majority of cases.

  Certain subgroups of patients have an increased incidence of lymphoma and Kaposi's sarcoma. There is currently no treatment or therapy for this disease.



  Epidemiological data as well as information regarding the types of patients who have contracted the disease suggest that an infectious agent transmitted through intimate contact may be the cause of the disease. Subsequently, three groups of researchers presented strong evidence indicating that the agent causing the AIDS is a retrovirus presenting a tropism directing it towards the T helper lymphocytes. These groups are:
 
   (a) RC Gallo and his collaborators at the National Institute of Health were able to isolate a cytopathic retrovirus (HTLV III) on patients presenting AIDS and pre-AIDS (RC Gallo et al., 1984, Science 224: 500; M. Popovic et al., 1984 (Science 224: 497) They also found antibodies to HTLV III in the serum of AIDS patients.
   (b) L.

  Montagnier and his collaborators isolated at the Institut Pasteur a lymphotropic retrovirus (LAV / HTLV III) on a patient who presented with cervical lymphadenopathy and also presented a risk of AIDS (Barre-Sinoussi, F. et al., 1983, Science 220 : 868). This group was also able to show the existence of antibodies against LAV / HTLV III in serum originating from patients suffering from AIDS (V.S. Kalyansraman et al., 1984, Science 225: 321). In addition, they were able to isolate LAV / HTLV III from the lymphocytes of a patient who had developed AIDS after receiving blood from an AIDS donor (PM Feorino et al., 1984, Science 225: 69) .
   (c) J. Levy and his collaborators have isolated infectious retroviruses (called AIDS-associated retroviruses (AIDS-associated retroviruses, or ARVs) from peripheral mononuclear cells of patients with AIDS (J.A.

  Levy et al., 1984, Science 225: 840).
 



  These three viruses have all been isolated independently, but they probably all belong to the same subgroup of retroviruses (J.A. Levy et al., 1984, Science 225: 840) and they will be collectively called LAV / HTLV III.



  The general structure of retroviruses is that of a ribonucleoprotein nucleus surrounded by an envelope containing lipids that the virus acquires during cell budding. The virally encoded glycoproteins are buried within the envelope and protrude outward. They determine the viruses known to the host and react with specific receptors located on the surface of sensitive cells. Neutralizing antibodies are thought to bind to the envelope glycoproteins and block their interaction with receptors located on the surface of cells (pages 534-535 in "The Molecular Biology of Tumor Viruses" tumor), published by John Tooze, 1973, Cold Spring Harbor Laboratory; pages 226-277 and 236-237 in "RNA Tumor Viruses" (RNA tumor virus (ribonucleic acid), published by R. Weiss, N. Teich, H.

  Varmus and J. Coffin, 1982, Cold Spring Harbor Laboratory). In the specific case of LAV / HTLV III, there is evidence that the T4 antigen, present in a subset of T lymphocytes, is the receptor or a component of the virus receptor (AG Dalgleish et al., 1984, Nature 312: 763; D. Kltazmann et al., 1984, Nature 312: 767).



  The LAV / HTLV III RNA genome is shown schematically in FIG. 1. Three genes are generally recognized: the gag gene codes for the internal structural proteins (nucleus proteins) of the virus and defines the antigens specific for the virus group. The pol gene codes for the reverse transcriptase associated with the virion. The env gene codes for viral glycoproteins. Other regions, marked sor and 3 min -orf, designate areas of the genome containing free-reading frames; the function of these regions is not currently known.



  A number of methods are currently used for the prevention and treatment of viral infections. They include vaccines that trigger an active immune response, treatment with chemotherapy agents and treatment with interferon.



  Traditional routes of vaccine preparation include the use of inactivated or attenuated viruses. Inactivation of viruses makes them harmless as biological agents, but does not destroy their immunogenicity. Injecting these "killed" virus particles into a host will then trigger an immune response capable of neutralizing future infection with a living virus. However, a major concern regarding the use of killed vaccines (using inactivated viruses) cancerns the risk of non-inactivation of all virus particles. Even if inactivation is carried out, since the killed viruses do not multiply in their host, the immunity obtained is often short-lived and additional immunizations are usually required. Ultimately, the inactivation process can alter viral proteins and make them less effective as immunogens.

  



  Mitigation refers to the production of strains of viruses that have essentially lost their ability to cause disease. One way to do this is to subject the virus to unusual growth conditions and / or frequent passage in cell culture. We then choose mutants of viruses that have lost virulence but are still capable of triggering an immune response. Attenuated viruses are generally good immunogens because they replicate in the host cell and trigger long-lasting immunity. However, there are several problems when using live vaccines, the most troublesome of which is insufficient attenuation.



   Instead of the above methods, one can use so-called subunit vaccines, or vaccine subunits. This implies an immunization obtained only using proteins containing the relevant immunological material. For many enveloped viruses, the virally encoded glycoprotein contains epitopes capable of triggering the production of neutralizing antibodies; they include La Crosse virus glycoproteins (F. Gonzalez-Scarano, RE Shope, CE Calisher and N. Nathanson, 1982, Virology 120: 42.), neonatal calf diarrhea virus (S. Matsuno and S. Inouye , 1983, Infection and Immunity 39: 155), the Venezuelan equine encephalomyelitis virus (JH Mathews and JT Roerhrig, 1982, J. Imm. 129: 2763), the Punta Toro virus (JM Dalrymple, CJ Peters, JF Smith and MK

  Gentry, 1981 in "Replication of Negative Strand Viruses", D.H.L. Bishop and RW Compans, page 167, Elsevier, New York), mouse leukemia virus (RA Steeves, M. Strand and JT August, 1974, J. Virol. 14: 187) and "Mouse Mammary Tumor Virus" ( mouse udder tumor virus) (RJ Massey and G. Schochetman, 1981, Virology 115: 20). An advantage of subunit vaccines is that uninteresting viral material is excluded.



  Vaccines are often given in combination with various adjuvants. Adjuvants help achieve a more durable and higher level of immunity, by using lower amounts of antigens in lower doses than if the immunogen was administered alone. The mechanism of action of adjuvants is complex and not fully understood. However, it may involve stimulation of phagocytosis and other activities of the reticuloendothelial system as well as delay in the release and degradation of the antigen. Examples of adjuvants include Freund's adjuvant (complete or incomplete), adjuvant 65 (containing peanut oil, mannide monooleate and aluminum monostearate), polyol "Pluronic L-121 "," Avridine ", and mineral gels such as aluminum hydroxide, aluminum phosphate or alum.

  Freund's adjuvant is no longer used in the formulation of vaccines for humans because it contains non-metabolizable mineral oil and is a potential carcinogen.



  The use of recombinant DNA technology for the production of subunit vaccines involves molecular cloning and expression in an appropriate vector of viral genetic information encoding proteins that can trigger a neutralizing response in the host animal. All other genetic information from the virus is excluded and only the proteins necessary to establish a neutralizing response are presented to the host animal. The host is never exposed to the whole virus and there is no risk of it becoming infected.



  Recently, an approach has been described which is potentially useful for the production of subunit vaccines (M. Mackett, GL Smith and B. Moss, 1982, Proc. Nat'1. Acad. Sci. 79, 7415-7419 ; M. Mackett, GL Smith and B. Moss, 1984, J. Virol. 49, 857-864; D. Panicali and E. Paoletti, 1982, Proc. Nat'1. Acad. Sci. 79, 4927-4931) . This approach involves the use of vaccinia virus as a vector to express foreign genes inserted into its genome. When introduced into animal hosts, the recombinant vaccinia virus expresses the inserted foreign gene and thus triggers a host immune response to such genes. Since the recombinant vaccinia viral virus can serve as a vaccine, such an approach combines the advantage of subunit (s) vaccines and live vaccines.



  The vaccinia virus contains a linear double-stranded DNA (deoxyribonucleic acid) genome with approximately 167 kilopairs of bases and it replicates within the cytoplasm of infected cells. These viruses contain a complete system of transcription enzymes (which includes styling, methylation and polyadenylation enzymes) within the nucleus of the virus, which are necessary for the infectivity of the virus. The vaccinia virus transcriptional regulatory sequences (promoters) allow transcription to be initiated by vaccinia RNA polymerase, but not by host cell RNA polymerase.



  Expression of foreign DNA in recombinant vaccinia viruses requires ligation of vaccinia promoters to DNA sequences encoding proteins of the foreign gene. Plasmid vectors, also called insertion vectors, have been constructed to insert chimeric genes into vaccinia viruses.

  One type of insertion vector is composed of: (a) a vaccinia virus promoter comprising the transcription initiation site; (b) several cloning sites by single restriction endonuclease, located downstream of the transcription start site for the insertion of fragments of foreign DNA; (c) vaccinia virus non-essential DNA (such as the TK gene) surrounding the promoter and cloning sites which direct the insertion of the chimeric gene into the non-essential homologous region of the virus genome; and (d) a marker of bacterial origin of replication and resistance to antibiotics for replication and selection in E. coli. Examples of such vectors are described by Mackett 2 (M. Mackett, J.L. Smith and B. Moss, 1984, J. Virol. 49, 857-864).



  Recombinant vaccinia viruses are produced by transfection of insertion plasmids of recombinant bacteria, containing the foreign gene, to cells previously infected with the vaccinia virus. Homologous recombination occurs within infected cells and produces the insertion of the foreign gene into the viral genome. Infected cells can be screened using immunological techniques, DNA plate hybridization, or genetic selection of recombinant viruses that can be isolated later. These vaccinia recombinants retain their essential functions and their infectivity and they can be constructed so as to contain approximately 35 kilobases of foreign DNA.



   The expression of foreign genes can be detected by enzymatic or immunological assays (for example by immunoprecipitation, radioimmunological assay or immunocoagulation). Natural membrane glycoproteins produced by cells infected with recombinant vaccinia are glycosylated and can be transported to the surface of cells. High levels of expression can be achieved by using strong promoters or by cloning multiple copies of a single gene.



  Recently, an approach has been described which is potentially useful for the production of recombinant proteins (Pennock et al., 1984, mol. Cel. Biol. 4: 399, Smith et al. 1983, J. Virol. 46: 584). This approach involves the use of a baculovirus type vector to express foreign genes inserted into its genome. After introduction into a cell of an appropriate host, the recombinant baculovirus expresses the foreign gene.



  The prototype of the baculovirus family is Auto-grapha californica, nuclear polyhedrosis virus (AcNPV). The genome of AcNPV consists of a species of circular double-stranded DNA of 128 kilobase pairs, which has been mapped against several restriction sites (G.E. Smith and M.D. Summers, 1978, Virology 89: 517). The virus replicates in the nucleus of infected insect cells. Two forms of virus are produced as a result of infection, by wild-type AcNPV, of sensitive cells, particles of virus with and without occlusion. Particles of virus in occlusion are surrounded by a protein called polyhedron whose coding is due to the polyhedrin gene (see Virol. 131: 561-565, 1983). In infected cells, occluded viral particles can be easily visualized using an optical microscope.



  Expression of foreign DNA in recombinant baculoviruses requires the ligation of baculovirus promoters on proteins encoding DNA sequences of the foreign gene. Vectors of the plasmid type, also called insertion vectors, have been constructed in order to insert chimeric genes into AcNPV.

  One type of insertion vector is composed of: (a) an AcNPV promoter comprising the transcription initiation site; (b) several single restriction endonuclease cloning sites, located downstream of the transcription start site for the insertion of foreign DNA fragments; (c) nonessential AcNPV DNA (such as the polyhedrin gene) flanking the promoter and the cloning sites which direct the insertion of the chimeric gene into the nonessential homologous region of the virus genome; and (d) a bacterial origin of replication and an antibiotic resistance marker for replication and selection in E. coli. Examples of such vectors are described by Miyamota et al. (1985, Mol. Cell. Biol. 5: 2860).



  Recombinant baculoviruses are reproduced by cotransfection of cells with recombinant bacterial insertion of plasmids containing the gene, with baculovirus DNA. Homologous recombination occurs within infected cells and causes the insertion of the foreign gene into the viral genome. Once a foreign gene is inserted into the polyhedrin gene, the occluded virus particles can no longer be produced and the resulting recombinant plaques can be visually examined for the absence of the occluded bodies. Infected cells can also be detected using immunological techniques, hybridization of DNA plaques, or genetic selection giving recombinant viruses that can be isolated later. These recombinant baculoviruses retain their essential functions and infectivity.



  The expression of foreign genes can be detected by enzymatic or immunological assays (for example by immunoprecipitation, radioimmunoassay or immunocoagulation). High levels of expression can be achieved by using strong promoters or by cloning multiple copies of a single gene.



  We will describe viruses which direct the expression of peptides or proteins related to epitopes of LAV / HTLV III. The recombinant viruses of the present invention, which express to LAV / HTLV III related epitopes eliciting an immunoprotective response, may be formulated as vaccines containing viruses to protect humans against infection due to LAV / HTLV III. In a particular embodiment of the present invention, live virus vaccine formulations can be prepared, using infectious viruses which express such LAV / HTLV III related epitopes in an infected host, but do not cause such disease in this host.



  The invention also includes peptides or proteins apparent to the LAV / HTLV III epitopes. Peptides or proteins that elicit an immunoprotection response can be formulated into subunit vaccines or multivalent vaccines to protect humans against LAV / HTLV III infection. Alternatively, the peptides or proteins of the invention may be used for diagnostic tests and assays for screening for AIDS or LAS.

  The peptides or proteins of the present invention can be produced in any expression vector system in host cells and isolated from this system; this includes, for example, cultures of animal cells or insects infected with the appropriate recombinant virus; microorganisms such as bacteria transfected with recombinant plasmids, cosmids or phages; and yeast transformed with recombinant plasmids. The present invention also provides methods, procedures and DNA constructs for the expression of genetic information encoding the LAV / HTLV III epitopes. Alternatively, the chemical synthesis of the peptides and proteins of the present invention can be carried out.



  In specific embodiments of the present invention, gene sequences encoding the envelope glycoproteins or the core structure proteins of LAV / HTLV III are inserted into plasmids (i.e. the LAV / HTLV III env or gag gene sequences, respectively), so that an anterior vaccinia virus promoter is placed in 5 min relative to the priming methionine sequence (ATG) of the LAV gene sequences / HTLV III, which gives the construction of chimeric genes flanked or surrounded by sequences of thymidine kinase (TK) of vaccinia DNA. These plasmids were transfected into cells which were previously infected with wild-type vaccinia virus, thus leaving the env gene or chimeric LAV / HTLV III gag, flanked by the TK sequences, to recombine in the gene. TK of vaccinia virus.

   The cells are allowed to lyse and the resulting viruses are cultured on TK cell plates <->. Recombinant viruses are chosen based on their ability to bind to cultures of these plaque cells in the presence of 5-bromo-deoxyuridine as well as by DNA-DNA hybridization on an appropriate radiolabeled probe comprising LAV envelope sequences / HTLV III or LAV / HTLV III gag. Positive plaques were purified for hybridization, challenged to expand, and resulting recombinant viruses tested for their ability to produce LAV / HTLV III envelope glycoproteins or LAV / HTLV III core structure proteins.

  LAV / HTLV III envelope-related proteins expressed by recombinant vaccinia viruses have been shown to be antigenic and immunogenic, and capable of eliciting both humoral and cellular mediated immunity in subhuman primates. The LAV / HTLV III gag-like proteins expressed by the recombinant vaccinia viruses were immunoreactive proteins that contained the major epitopes of authentic nucleus proteins.



  In other specific embodiments included in the present invention, LAV / HTLV III env or gag sequences were inserted into a plasmid such that a baculovirus polyhedrin promoter was placed in the 5 min position relative to the priming methionine (ATG) sequence of the LAV / HTLV III gene followed by polyhedrin DNA sequences at the 3 min end. These plasmids were then co-transfected into cells with wild-type baculovirus DNA, allowing the chimeric LAV / HTLV III gene flanked by additional sequences of AcNPV to recombine in the polyhedrin gene of baculovirus. The cells were allowed to lyse and the resulting viruses were cultured on plates.

  By visual examination to detect the lack of occluded bodies or by DNA-DNA hybridization on env / HTLV III or LAV / HTLV III radiolabelled probes, recombinant viruses were selected. Recombinant plaques were purified and propagated, and the resulting recombinant viruses screened for the ability to produce LAV / HTLV III related proteins by immunoprecipitation followed by analysis on SDS (sodium dodecylsulfate) -polyacrylamide gels. Lysates of infected cells were tested for the ability to detect LAV / HTLV III antibodies in serum by ELISA.



  We will now briefly describe the attached figures.



  Figure 1 shows the integrated proviral genome structure of LAV / HTLV III. The hatched areas indicate regions of open reading frames. The regions coding for the group-specific antigen, reverse transcriptase and envelope proteins are designated respectively by gag (group-specific antigen), pol and env. Overlapping open reading frames are represented by cross-hatched areas. The numbers designate the numbers of base pairs downstream of the styling site ("cap" site), where viral transcription begins. Restriction sites are marked as follows: Bg, Bg1II; Ec, EcoRI; Hn, HindIII; kp, kpnI; Ss, SstI.



  FIG. 2 represents the nucleotide sequence of the specific region of LAV / HTLV III (EcoRi to SstI) present in the plasmid pRS-3 DNA; the entire LAV / HTLV III envelope gene (nucleotide 5766 to 8349) is contained in the LAV / HTLV III insert. The restriction sites used in the construction of pv-envl, pv-env2 and pv-env5 are indicated. The entire amino acid sequence of the envelope gene, as derived from the nucleotide sequence data, is also shown.



  FIG. 3 is a schematic representation of the construction of plasmids containing a portion of the sequence coding for LAV / HTLV III envelope proteins, a sequence inserted downstream of a vaccinia virus promoter. The sequence encoding the LAV / HTLV III envelope is represented by the free bar and the vaccinia promoter by the shaded or hatched bar. The nucleotide sequence at the junction of the vaccinia promoter region and the coding sequence for the LAV / HTLV III envelope is shown in the lower part of the figure. The underlined sequences indicate the presumed priming codons and the reading frame for the chimeric gene. Note that the third and fourth amino acids of the translated sequence (Pro-Val) of the recombinant pv-env2 correspond to amino acids number 43 and 44 of the envelope coding sequence of LAV / HTLV III.



  Figure 4 is a schematic representation of the construction of plasmids containing the entire coding sequence of the LAV / HTLV III envelope proteins inserted downstream of a vaccinia virus promoter. The vaccinia virus promoter is represented by the hatched or shaded bar. Plasmid pv-env5 containing the entire LAV / HTLV III envelope coding region was constructed in two steps. The 5 min and 3 min portions of the coding region were first cloned separately in pGS20 to form pv-env1 which contains the amino coding end of the LAV / HTLV III envelope gene and pv-env2 which contains the carboxyl terminus coding for the LAV / HTLV III envelope gene. These two portions are grouped together at the StuI site as shown.



  Figure 5 is a schematic representation of the construction of plasmids containing the coding sequence of the LAV / HTLV III envelope proteins, without the transmembrane (anchor) sequence, inserted downstream of a vaccinia virus promoter. The plasmid pv-env7 was constructed in two stages. The 5 min part of pv-env 5 was inserted into p26 to form an intermediate plasmid, pv-env5 / 26, which provided a frame translation termination sequence (TAA) giving a truncated env gene. The truncated env sequence, terminated by TAA, was then inserted into pG20 in order to construct pv-env7 in which the truncated env gene is flanked by sequences of the vaccinia TK gene.



   FIG. 6 represents the construction and the choice of the recombinant vaccinia virus. The solid bars represent the thymidine kinase (TK) gene of the vaccinia virus. This TK gene is also present in the plasmid pv-env5 DNA, but in the plasmid the TK gene is interrupted by the chimeric gene consisting of the 7.5K vaccinia promoter (hatched bar) and by the coding region of LAV / HTLV III envelope (open bar). After infection of the cells with vaccinia, the recombinant plasmid containing the TK gene interrupted by the envelope gene of LAV / HTLV III is introduced into the infected cells. Recombinations that occur in the TK sequences surrounding the chemical gene introduce the envelope gene sequence of LAV / HTLV III into the genome of vaccinia virus. The resulting recombinant virus, containing the LAV / HTLV III envelope gene, is TK.



  FIGS. 7 represent a characterization of the genome structures of the recombinant vaccinia viruses containing the env gene of a LAV / HTLV III. FIG. 7A represents an analysis, using a restriction enzyme, of the vaccinia recombinants v-env5 and v-env5NY as well as their respective parental vaccinia strains (WR and v-NY, respectively). DNA fragments generated by HindIII restriction enzyme digests were resolved by electrophoresis on an agarose gel and stained with ethidium bromide to visualize the bands. FIG. 7B represents the Southern clot obtained after transfer of the resolved fragments by restriction on nitrocellulose and hybridization on a throttle translation probe, specific for the LAV / HTLV III envelope sequences.



  FIG. 8A represents an analysis by Western immunocoagulation ("west") of the proteins produced by the recombinants of env-LAV / HTLV III of vaccinia. Proteins from the following sources were resolved on a gradient polyacrylamide gel of 7 to 15% SDS and electrotransfered onto nitrocellulose paper: LAV / HTLV III virion proteins (LAV / HTLV III); cells infected with wild-type vaccinia virus ("WTvv"); ("Wild-type vaccinia virus"); uninfected cells ("mock"); cells infected with recombinant v-env2 (v-env2) or v-env5 (v-env5) viruses. Proteins immunorative to the serum of an AIDS patient have been detected by the action of a peroxidase conjugated to anti-human IgG antibodies. The LAV / env genes produced are indicated by gp150, gp110 and gp41.

  Molecular weight standards are expressed in kilodaltons (kd).



  FIG. 8B represents an analysis of a Western immunocoagulate of proteins produced by recombinant vaccinia LAV-env in two types of cells. Proteins from BSC-40 cells or HeLa cells were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to nitrocellulose paper, then reacted with pooled serum from people serum positive for LAV / HTLV-III; lanes 1 and 5 represent the cells infected with "mock"; lanes 2 and 6 represent cells infected with the wild-type vaccinia virus; lanes 3 and 7 represent cells infected with v-env5; and lanes 4 and 8 represent cells infected with v-env2. Immunoreactive proteins were detected using protein A labeled with <1> <2> <5> I.

  LAV-env gene type products are indicated by gp150, gp110 and gp41.



  FIG. 9A represents the results of a radioimmunoprecipitation analysis of the proteins synthesized in cells infected by the wild-type vaccinia virus ("WTvv") or by recombinant vaccinia virus (v-env2 and v-env5). Proteins have been labeled with <3> <5> S-methionine 10 to 12 hours after infection. The labeled proteins present in cell lysates were reacted with human control serum (N) or with serum from an AIDS patient (I) and the immune complexes were precipitated by Staphylococcus protein A aureus. The immunoprecipitated proteins were resolved by electrophoresis on a polyacrylamide gel containing 15% SDS and detected by fluorography. Molecular weights are given in kilodaltons (kd).



  FIG. 9B represents the results of a radioimmunoprecipitation analysis of proteins labeled with 3H-glucosamine produced by the expression effect of the vaccinia-LAV / HTLV III recombinants of the invention. Hela cells were either "infected" with non-infectious cells ("mock"; noninfection) (tracks 1 and 5), or infected with wild-type vaccinia viruses (tracks 2 and 6), v-env5 (tracks 3 and 7) or v-env2 (tracks 4 and 8) and marked with <3> H-glucosamine. The cell lysates were immunoprecipitated with normal human serum (lanes 1-4) or with pooled serum from people serum positive for LAV / HTLV III (lanes 5-8) and protein A. The immunoprecipitated proteins were resolved by SDS-polyacrylamide gel electrophoresis.



  FIG. 9C represents the results of a “pulse-hunting” radioimmunoprecipitation analysis of the LAV / HTLV III envelope proteins produced by the vaccinia LAV / HTLV III recombinants. HeLa cells were infected with wild-type vaccinia virus, v-env5 or v-env2 as indicated, labeled with <3> <5> S-methionine, and we washed with a hunting medium. At the following time intervals, the cells were washed, lyzed and immunoprecipitated with pooled serum from persons serum positive for LAV / HTLV III and resolved by SDS-PAGE (electrophoresis on polyacrylamide gel): 0 hour (lanes 1, 7, 13); half an hour (tracks 2, 8, 14); 1 hour (tracks 3, 9, 15); 2 hours (tracks 4, 10, 16); 6 hours (tracks 5, 11, 17) and 12 hours (tracks 6, 12, 18).

  Lane M represents markers comprising molecular weight standards marked with <1> <4> C.



  FIG. 9D represents the results of a radioimmunoprecipitation analysis of proteins related to the envelope of LAV / HTLV III which are found in cells and in media originating from cells infected with vaccinia virus-LAV / HTLV III recombinants of the invention. HeLa cells were either "infected" with uninfected viruses (ie, uninfected; lane 1 "mock"), or infected with wild-type vaccinia virus (lane 2) v-env5 ( track 3) or v-env2 (track 4) and marked with <3> <5> S-methionine. The cells were separated from the medium and lysed. The cell lysates (pellet) and the upper supernatant were each immunoprecipitated using group serum from persons serum positive for LAV / HTLV III. The immunoprecipitated proteins were resolved by gel electrophoresis; polyacrylamide (SDS-PAGE).



   FIG. 9E represents the result of a radioimmunoprecipitation analysis of proteins related to the envelope of LAV / HTLV III in cells and in media originating from cells infected with recombinant vaccinia-LAV / HTLVL III viruses of the invention . Recombinant v-env5 contains the entire LAV / HTLV III envelope coding sequence, while v-env7 contains the LAV / HTLV III envelope coding sequence except the transmembrane region (anchoring). Hela cells were either "infected" with uninfected viruses (ie, uninfected; lane 1 "mock"), or infected with wild-type vaccinia virus (lane 2), v-env5 (track 3) or v-env2 (track 4), and marked with <3> <5> S-methionine. The cells were separated from the medium and lysed.

  The cell lysates (pellet) and the medium (supernatant) were each immunoprecipitated using pooled serum from persons serum positive for LAV / HTLV III. The immunoprecipitated proteins were resolved by polyacrylamide gel electrophoresis (SDS-PAGE).



  FIG. 10 represents a Western immunocoagulation analysis of serum samples from mice immunized with recombinant vaccinia-LAV / HTLV III viruses. The mice had been immunized with recombinant vaccinia virus v-env5 or v-env2. After 8 weeks, the serum samples were reacted with LAV / HTLV III virion proteins which had been resolved by electrophoresis on SDS-PAGE (polyacrylamide gel) and electrotransfered onto nitrocellulose paper. A goat anti-mouse immunoglobulin conjugated with alkaline phosphatase was used to detect the LAV / HTLV III proteins which were recognized by the sera of mice.

  Lanes a to e represent the serum samples from 5 individual mice inoculated with v-env5, and lanes f to k represent serum samples from 5 individual mice inoculated with v-env2. Pooled sera from LAV / HTLV III positive people (AIDS) and non-immune C57B16J (NMS) mice served as positive and negative controls, respectively. The positions of the LAV / HTLV III envelope glycoproteins gp150, gp110 and gp43 are indicated.



  FIG. 11 is a histogram representing the seroconversion of macaque monkeys vaccinated with recombinant vaccinia virus, v-env5. We inoculated 4 macaque monkeys, by scarification of the skin, 2 x 10 <8> 2 x 10 plate and 4 monkey training units <7> v-env-5 plate forming units. 2 x 10 animals were inoculated <7> vaccine-herpes simplex gD control recombinant plaque-forming units (v-HSVgD1). Ten weeks after the first inoculation, all animals except number 81 were administered a second 2 x 10 inoculation. <8> plaque forming units of the same virus. Serum samples were collected before inoculation (open bars); 4 weeks after the first inoculation (shaded bars; and 4 weeks after the second inoculation (solid bars).

  The sero-conversion was verified and titrated by ELISA, using purified LAV / HTLV III virions as target antigen.



  FIG. 12 represents a Western immunocoagulation analysis of serum samples from macaque monkeys immunized with recombinant vaccinia-LAV / HTLV III viruses, v-env5, as described for FIG. 11. Samples were collected. virus before the first inoculation (pre track) and after 4 weeks after the second inoculation. Aliquots of the serum were diluted 50-fold and reacted with LAV / HTLV V virion protein which was resolved by polyacrylamide gel chromatography (SDS-PAGE) and immobilized on nitrocellulose filters , by electrotransfer using a protocol which allows the optimal detection of the two envelope glycoproteins, gp110 (figure 12A) and gp41 (figure 12B).

  The proteins of LAV / HTLV III recognized by the macaque sera were detected by anti-human goat immunoglobulin conjugated with alkaline phosphatase. Pooled sera from people with positive serum were used as a positive control for LAV / HTLV III (AIDS) testing. Authentic LAV / HTLV III (AIDS) virions were used as a control.



  FIG. 13 represents an analysis by Western immunocoagulation of these serum samples coming from chimpanzees immunized by recombinant vaccinia viruses NY-LAV / HTLV III, v-env5NY. Two x 10 chimpanzees were inoculated intradermally <8> v-env5NY plaque-forming units, however, the same dose of v-HSVgD1NY, a vaccinia-herpes simplex gD virus recombinant constructed from the same vaccinia strain, was inoculated into an animal parental-NY. All animals received a second inoculation 8 weeks after the first inoculation. Serum samples were collected before immunization (lane 1), 8 weeks after primary immunization (lane 2) and 2 weeks after the second immunization (lane 3).

  Aliquots of serum were diluted 50-fold and reacted with LAV / HTLV III virion proteins which were resolved by polyacrylamide gel electrophoresis (SDS-PAGE) and immobilized on nitrocellulosic filters by electro- transfer, using a protocol which allows the best possible detection of gp 41. The proteins of LAV / HTLV III recognized by the chimpanzee sera were detected by anti-human goat immunoglobulin conjugated with alkaline phosphatase. Pooled sera from persons whose serum was positive for LAV / HTLV III (AIDS) were used as a positive control.



  FIGS. 14A to 14D represent the nucleotide sequence of the specific region of LAV / HTLV III (SstI to KpnI) present in the DNA of plasmid pKS-5; the entire LAV / HTLV III gag gene (nucleotides 340 to 1871) is contained in the inserted part of LAV / HTLV III. The restriction sites used for the construction of pAc-gag1 are indicated. The full amino acid sequences of the gag gene, as inferred from the nucleotide sequence information, are also shown.



   FIG. 15 is a schematic representation of the construction of the plasmid pAc-gag1 containing the coding sequence of the LAV / HTLV III gag proteins inserted downstream of an AcNPV promoter. The cloning vector pAc610 contains nucleotide sequences corresponding to the polyhedrin gene promoter and it includes the leader sequences in 5 min and the polyhedrin sequences in 3 min interrupted by cloning sites located downstream from the start of transcription site . The cloning sites at position -8 are EcoRI, SstI, SmaI (XmaI), BamHI, XbaI and PstI. SalI, AccI and HincII are not unique. There are no sites for KnpI or Bg1II.



  FIG. 16 schematically represents the construction and the selection of the recombinant AcNPV (Ac-gag1). The hatched bar indicates the polyhedrin promoter and leader sequences in 5 min. The solid bars represent the baculovirus polyhedrin gene. The parts of this gene are present in the plasmid pAc-gag1 DNA, interrupted by the coding region of LAV / HTLV III gag (open bar). Cells are co-transfected with recombinant recombinant plasmid DNA containing portions of the polyhedrin gene interrupted by the LAV / HTLV III gag gene (pAc-gag1) with wild type AcNPV DNA. Recombinations that occur in the polyhedrin sequences flanking the chimeric gene introduce the LAV / HTLV III gag gene sequence into the genome of AcNPV.



  Figure 17 shows a Northern (North) coagulation analysis of RNA extracted from Spodoptera frugiperda cells infected with wild-type AcPNV and Ac-gag1, using specific waves for LAV / HTLV III gag.



  FIG. 18 represents the result of a radioimmunoprecipitation analysis of the proteins expressed in cells infected with recombinant baculovirus Ac-gag1. Proteins have been marked with ( <3> <5> S) methionine, for 2 hours, 24 hours (tracks 1, 2), 48 hours (tracks 3, 4) and 72 hours (tracks 5, 6) after infection. The labeled proteins present in the cell lysates were reacted with human control serum (tracks 1, 3, 5) or with the serum of an AIDS patient (tracks 2, 4, 6), and the immune complexes were precipitated by protein A of Staphylococcus aureus. The immunoprecipitated proteins were resolved by electrophoresis on a 10% SDS polyacrylamide gel and detected by fluorography. Molecular weights are given in kilodaltons.



  FIG. 19 represents the results of a “pulse-hunting” radioimmunoprecipitation analysis of proteins related to LAV / HTLV III gag which are found in cells infected with the recombinant AcNPV of the invention. Spodoptera frugiperda (Sf9) cells were infected with Ac-gag1 and pulsed for 5 minutes to 24 hours after infection. The cells were then washed with complete medium and incubated with complete medium for 0 hours (tracks 1, 2), 2 hours (tracks 3, 4), 4 hours (tracks 5, 6) and 8 hours ( tracks 7, 8). At the indicated times, the cells were washed, lysed and immunoprecipitated with pooled serum from people seropositive for LAV / HTLV III (tracks 2, 4, 6, 8) or normal human serum (tracks 1, 3, 5, 7).

  The proteins were resolved by SDS-polyacrylamide gel electrophoresis.



  FIG. 20 schematically represents the construction of five plasmids, pv-gag1, pv-gag2, pv-gag3, pv-gag4 and pv-gag5 containing the 7.5K vaccinia virus promoter ligated to various lengths of the LAV genome / HTLV III containing gag coding sequences. The cloning vector pGS62 which is used in these constructions is identical to pGS20 (see FIG. 3), except that it comprises a single EcoRI site located downstream of the unique SmaI site of pGS20.



  FIG. 21 represents the results of an analysis of Western type (West) immunocoagulation of the proteins expressed in cells infected by recombinant LAV / HTLV III vaccinia viruses (v-gag1NY, v-gag2NY, v-gag3NY, v -gag4NY and v-gag5NY), the starting vaccinia virus (v-NY) and cells "infected" with non-infectious cells ("MOCK"). Samples of purified LAV / HTLV III virions ("LAV / HTLV III") were included as positive controls. BSC-40 confluent cells were infected at a multiplicity of infection index of 10. The infection was allowed to continue for 12 hours, after which the cells were harvested and lysed. Total cell proteins were resolved by SDS-PAGE gel electrophoresis and immobilized by electrotransfer on nitrocellulose.

  The filters were reacted with (1) serum from an AIDS patient (Figure 21B) which was detected using an anti-human goat immunoglobulin conjugated with alkaline phosphatase; (2) mouse monoclonal antibodies which define gag proteins p25 and p18 (Figures 21A and 21C), which were then detected using goat anti-mouse immunoglobulin conjugated with alkaline phosphatase.



  FIG. 22 schematically represents the construction of the plasmid pAc-env5 containing the entire coding sequence of the LAV / HTLV III envelope inserted downstream of a polyhedrin promoter AcNPV. The cloning vector pAc610 is described in Figure 15 above.



  FIG. 23 represents the results of a radioimmunoprecipitation analysis of the proteins expressed in cells infected with the recombinant baculovirus Ac-env5. Cells "infected" with non-infectious viruses ("MOCK") and the starting baculovirus strain (AcNPV) are included as controls. The proteins expressed by infected Sf9 cells were labeled, 28 hours after infection, for 2 hours by <3> <5> S-methionine. The labeled proteins present in cell lysates were reacted with AIDS patient serum or with mouse monoclonal antibodies which define gp110 or gp41, and the immune complexes were precipitated using protein A of Staphylococcus aureus. The immunoprecipitated proteins were resolved by SDS-PAGE gel electrophoresis and the immunoprecipitated proteins were detected.



   We will now present a more detailed description of the invention.



  The subject of the invention is a recombinant virus according to claim 1.



  The present invention also includes a method of obtaining viruses which produce peptides and proteins related to LAV / HTLV III epitopes. The invention also relates to peptides and proteins related to LAV / HTLV III epitopes, which can be produced using methods using recombinant DNA or by chemical synthesis. Viruses or peptides and proteins of the present invention which can elicit an immune protective response can serve as immunogens in a variety of vaccine formulations, including multi-purpose vaccine formulations, to protect against infection by LAV / HTLV III, which is the agent responsible for LAS (lymphadenopathy syndrome) and AIDS.

  As a variant, the peptides or proteins included in the invention can be used as antigens in diagnostic immunoassays or assays for the detection, in a patient, of specific antibodies of LAV / HTLV III. The peptides or proteins which are used in the immunological tests or assays included in the invention must be antigenic (that is to say capable of reacting with antibodies of a patient concerning LAV / HTLV III), but they do not need to be immunogenic (i.e. capable of eliciting an immune response). Furthermore, the antigens used in the diagnostic immunoassay or assay do not have to elicit an immune protective response in vivo.



  According to one embodiment included in the present invention, techniques using recombinant DNA are used to insert nucleotide sequences encoding LAV / HTLV III epitopes into expression vectors which will direct the expression of LAV / HTLV III sequences in appropriate host cells. These expression vector systems-host cells can be used to produce in vitro peptides and proteins related to LAV / HTLV III and, in this case, the genes produced can be purified from the cells in culture. Peptides or proteins that can elicit an immune protective response and serve as an immunogen in subunit vaccine formulations.

  Alternatively, peptides and proteins, immunogenic or simply antigenic, of the invention may be used as antigens in immunoassays and assays designed to detect antibodies from specific LAV / HTLV III patients. In another approach for the production of the peptides and proteins of the invention, the amino acid sequences of these peptides and proteins can be deduced from the nucleotide sequences of LAV / HTLV III contained in the recombinants which express antigenic peptides and proteins and / or LAV / HTLV III related immunogens. These peptides and proteins can then be the subject of chemical syntheses and serve in vaccine formulations with synthetic subunits (if they are immunogenic) or as antigens in immunological tests or assays for diagnostics (if they are antigenic and / or immunogenic).



  When the expression vector is a virus which directs the expression of an immunogen linked to an LAV / HTLVIII epitope capable of eliciting an immune protective response, the virus itself can be formulated as a vaccine. Infectious recombinant viruses, which do not cause disease in the host, can be used in virus vaccine preparations to confer significant immunity. Alternatively, vaccines containing inactivated viruses can be prepared when "killed" viruses are used. In addition, multivalent vaccines containing LAV / HTLV III epitopes as well as those of other disease-causing agents can be prepared.



  For the sole purpose of presenting a clear description, the method included in the invention can be divided into the following stages: (a) isolation of a gene, or gene fragment, encoding proteins of the LAV / HTLV III virus; (b) insertion of the gene, or of the gene fragment, into expression vectors; (c) identifying and growing the recombinant expression vector in a host system which is capable of replicating and expressing the gene; (d) identification and purification of the product obtained using the gene; (e) determination of the immune power of the product, and (f) formulation of a vaccine.



  In specific embodiments included in the invention, there is described the construction of recombinant vaccinia viruses and baculoviruses containing the LAV / HTLV III envelope gene which directs the expression of proteins immunologically linked to proteins of LAV / HTLV III envelope in tissue culture cells infected with recombinant viruses. In other embodiments included in the present invention, there is described the construction of recombinant vaccinia viruses and baculoviruses containing the LAV / HTLV III gag gene which directs the expression of proteins immunologically related to structural proteins of nuclei of LAV / HTLV III in culture cells of these tissues infected with recombinant viruses.

  However, the compositions and methods described herein are not limited to the construction of recombinant viruses expressing envelope-related proteins or LAV / HTLV III gag, and they can be used to construct recombinants in n any expression vector system for the production of polypeptides related to antigens of any ethilogic agent of AIDS.



  For the sake of clarity, the entire process will be studied in terms of the envelope and gag genes of LAV / HTLV III. The same technique, however, can be applied analogously to construct recombinant expression vectors and to produce polypeptides related to any of the LAV / HTLV III proteins as well as those of related viruses. Such proteins include, without being limiting, the products of the LAV / HTLV III genes, such as gag, pol and env and at least four additional genes called to date: sor, tat, 3 min -orf and a gene variously called art or very (see Fisher et al, 1986, Science 233: 655-659).


 ISOLATION OF GENES, OR GENE FRAGMENTS, ENCODING LAV / HTLV III VIRAL PROTEINS
 



   The isolation of the LAV / HTLV III genes first involves the isolation of DNA fragments containing the envelope gene sequences. As previously explained, LAV / HTLV III has an RNA genome and, therefore, one can obtain the corresponding DNA which codes for the LAV / HTLV III gene (a) by cloning by cDNA the RNA isolated from purified LAV / HTLV virions. III, or (b) by cloning cDNA from poly-containing RNA <SEP> A <SEP> which is obtained from cells infected with LAV / HTLV III, or (c) by cloning purified genome DNA from cells infected with LAV / HTLV III. Hereinafter, the DNA coding for the LAV / HTLV III genes will be called "LAV / HTLV III DNA".



  In order to generate LAV / HTLVIII DNA fragments, LAV / HTLV III DNA can be cleaved at specific sites, using various restriction enzymes. Alternatively, ase DNA can be used in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, for example by ultrasound treatment. The linear DNA fragments can then be separated, according to their dimensions, by conventional techniques, which includes, without being limiting, electrophoresis on agar and polyacrylamide gel and column chromatography.



  Any restriction enzyme or combination of restriction enzymes may be used to generate one or more fragments of LAV / HTLV III DNA containing the gag or envelope sequences, provided that the enzymes do not do not destroy the antigenicity of the gene protein product. For example, the antigenic site of a protein can consist of about 7 to about 14 amino acids. Thus, a protein the size of the envelope precursor peptide (approximately 97,000 daltons) can have many separate antigenic sites, perhaps thousands considering the overlapping sequences, considerations of secondary and tertiary structures and possibilities of treatment like acetylation, glycosylation or phosphorylation.

  Therefore, many partial sequences of envelope polypeptide genes may code for an antigenic site. Therefore, many combinations of restriction enzymes can be used to generate DNA fragments which, when inserted into an appropriate vector, are capable of directing the production of envelope specific amino acid sequences comprising different antigenic determinants .



  Once the DNA fragments have been generated, identification of the specific DNA fragment containing the LAV / HTLV III envelope gene can be carried out in a number of ways. First, it is possible to sequence the DNA fragments corresponding to the entire LAV / HTLV III genome, then to identify the fragments containing the sequence of envelope protein genes or of gag gene based on a comparison. the predicted amino acid sequence and the amino acid sequence of the envelope protein or gag nucleus proteins. Second, once the entire genome sequence has been determined, large open reading frames can be ordered from 5 min to 3 min.

  As the genome organization of all the retroviruses examined so far is 5 min -gag-pol-env-3 min, the large open reading frame closest to the 3 min end is very likely to code for the envelope gene, while the large open reading frame closest to the 5 'end is most likely to encode the gag gene. Third, probable identification of a specific gene can be made by recognizing homology with other known retrovirus genes, by nucleic acid hybridization analyzes or sequence comparisons if the sequences are known. .



  Alternatively, the fragment containing the envelope protein gene can be identified by selection of mRNAs. In this procedure, the LAV / HTLV III DNA fragments are used to isolate, by hybridization, the complementary mRNAs. An immunoprecipitation analysis of the in vitro translation products of the isolated mRNAs identifies the mRNA and therefore, the complementary LAV / HTLV III DNA fragments which contain the sequences of the envelope proteins. Finally, one can choose specific mRNAs of envelope proteins by adsorption of polysomes isolated from cells infected with LAV / HTLV III on immobilized antibodies directed against the envelope or gag proteins. One can synthesize a radiolabelled envelope DNA, ADNC (complementary DNA) using as template the chosen mRNA (coming from the adsorbed polysomes).

  The radiolabelled mRNA or the radiolabelled cDNA can then be used as a probe to identify the DNA fragments of LAV / HTLV III containing sequences of envelope or gag genes. Instead of isolating the envelope or gag gene, it is possible, without this list being exhaustive, to carry out chemical synthesis of the gene sequence itself (provided that the sequence is known) or to transform cDNA into MRNA that codes for the envelope or gag gene.



  Once identified and isolated, the LAV / HTLV III DNA fragment containing the sequences of interest can first be inserted into a cloning vector, such as a plasmid cloning sector, which is used to transform appropriate host cells. in order to cause DNA replication so as to generate numerous copies of the interesting sequences of LAV / HTLV III. This can be achieved by ligation of the LAV / HTLV III DNA fragment into a cloning vector with complementary cohesive terminations. However, if the complementary restriction sites used to fragment the LAV / HTLV III DNA are not present in the cloning vector, the ends of the DNA molecules may be modified.

  Such modifications include the production of blunt ends by digestion of the single stranded DNA terminations or by packing the single stranded terminations so that the ends can undergo blunt end ligation. Alternatively, any desired site can be produced by ligating nucleotide sequences (binding agents) to the DNA termini; these ligated binding agents can comprise specific oligonucleotides, synthesized chemically, and encoding restriction site recognition sequences. According to other methods, the cleaved vector and the DNA fragment of LAV / HTLV III can be modified, by homopolymer termination.



   The transformation of host cells by recombinant DNA molecules which incorporate the isolated gene, by cDNA or a DNA sequence obtained by synthesis, makes it possible to generate multiple copies of the gene. Thus, the gene can be obtained in large quantities by the growth of transformants, the isolation of the recombinant DNA molecules from the transformants and, when necessary, the recovery of the inserted gene from the isolated recombinant DNA.



  If the ultimate goal is to insert the gene into virus expression vectors, such as vaccinia virus or adenovirus, the recombinant DNA molecule that incorporates the LAV / HTLV III gene can be modified so that the gene is surrounded by virus sequences allowing genetic recombination in virus infected cells, so that the gene can be inserted into the viral genome.



  The entire LAV / HTLV III genome was cloned and sequenced by Wain-Hobson et al. (S. Wain-Hobson et al., 1985, Cell 40: 9). One clone, called lambda J19, contained a 9.2 kilopair base DNA fragment from a LAV genome sequence inserted into the Hind III site of lambda L 47.1.



  A particularly useful lambda J19 subclone containing the LAV / HTLV III envelope gene is pRS-3, which consists of a fragment, at 3840 EcoRI verses SstI base pairs, of the nucleotide sequence of LAV / HTLV III inserted into the EcoRI and SstI site of pUC18. The specific DNA of LAV / HTLV III contained in pRS-3 is located between the EcoRI site placed at nucleotide 5289 and the SstI site placed at nucleotide 9129 on the LAV / HTLV III genome (S. Wain-Hobson et al., 1985, Cell 40: 9); see Figure 2 which shows the nucleotide sequence of LAV / HTLV III contained in pRS-3. However, due to the degeneracy of the sequences encoding the nucleotides, it is possible to use in the practice of the present invention, for the cloning of the envelope gene of LAV / HTLV III, other DNA sequences which encode practically the same amino acid sequence as shown in Figure 2.

  This includes, but is not limited to, nucleotide sequences comprising all or parts of the envelope nucleotide sequence shown in Figure 2, which are modified by replacement of different codons which encode the same amino acid residue or a residue functionally equivalent amino acid within the sequence (for example an amino acid of the same polarity), producing a silent change.



  Particularly useful subclones of lambda J19 containing the LAV / HTLV III gag gene are pKS-5 and pSS5. The plasmid pKS5 consists of a fragment of LAV / HTLV III comprising 3148 base pairs from SstI to KpnI in pUC18. The specific DNA of LAV / HTLV III contained in pKS-5 goes from the SstI site located on nucleotide 224 to the KpnI site located at nucleotide 3372 on the LAV / HTLV III genome. The plasmid pSS-5 consists of a 5.1 kbp fragment of SstI to SalI of LAV / HTLV III in pUC 18. The specific DNA of LAV / HTLV III contained in pSS-5 comes from the SstI site located at nucleotide 224 up to 'at the SalI site located at nucleotide 5331 on the LAV / HTLV III genome. (Wain-Hobson, S. et al., 1985, Cell 40: 9), see Figure 14, which describes the nucleotide sequence of LAV / HTLV III contained in pKS-5 and PSS-5.

  However, due to the degeneracy of the nucleotide coding sequences, the practice of the present invention can be used for the cloning of the LAV / HTLV III gag gene, other DNA sequences which encode substantially the same amino acid sequence than that shown in Figure 14. This includes, but is not limited to, nucleotide sequences comprising all or parts of the gag nucleotide sequence shown in Figure 14, which are modified by substitution of different codons which encode the same amino acid residue, or a functionally equivalent amino acid residue, in the sequence (for example an amino acid of the same polarity), thereby producing a silent change.


 INSERTION OF LAV / HTLV III PROTEIN CODING SEQUENCES IN EXPRESSION VECTORS
 



  The nucleotide sequence coding for the envelope protein of LAV / HTLV III, or a part of this sequence, is inserted into an appropriate expression vector, that is to say a vector which contains the elements necessary for the transcription and translation of the coding sequence of inserted proteins. Various host-vector systems can be used to express the coding sequence of proteins. This includes, but is not limited to, mammalian cell systems infected with viruses (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with viruses (e.g. baculovirus); microorganisms such as yeast containing yeast vectors or bacteria transformed by bacteriophage DNA, plasmid DNA or cosmid DNA.

  The elements of expression of these vectors vary in strength and specificity. Depending on the host-vector system used, any one of a number of suitable transcription and translation elements can be used. For example, when cloning into mammalian cell systems, promoters isolated from the genome of mammalian cells (for example a mouse metallothioniene type promoter) or isolated from viruses whose growth can be used takes place in these cells (for example the 7.5K promoter of vaccinia virus). Promoters produced by recombinant DNA or by synthetic techniques can also be used to ensure transcription of the inserted sequences.



  Specific priming signals are also required for efficient translation of the inserted protein coding sequences. These signals include the ATG boot codon and adjacent sequences. When the entire LAV / HTLV III envelope gene or gag, including its own start codon and adjacent sequences, is inserted into the appropriate expression vectors, additional control signals for translation may not be required. However, when inserting only part of the envelope coding sequence, or gag, it is necessary to provide exogenous translation control signals, which includes the initiation codon or ATG translation initiation.

   The priming or initiation codon must also be in phase with the reading frame of the coding sequences of envelope proteins to guarantee the translation of the entire inserted sequence. These exogenous translation control signals and these initiation or translation initiation codons can have various origins, both natural and synthetic.



  Any of the previously described methods for inserting DNA fragments into a vector can be used to construct expression vectors containing a chimeric gene consisting of appropriate transcription / translation control signals and the coding sequences of proteins. These methods may include techniques using "in vitro" recombinant DNA and synthetic techniques and "in vivo" recombination (genetic recombination).



  In the particular embodiments described in detail in the examples of the present invention, the vaccinia virus and the baculovirus were chosen as expression vectors. However, the invention is not limited to the use of vaccinia virus or baculovirus. As previously explained, the expression vectors which can be used include, without being limited to the following vectors or their derivatives: viruses infecting humans or animals such as vaccinia virus or adenoviruses; viruses infecting insects such as baculoviruses; yeast-like vectors; vectors of the bacteriophage type, and vectors of the plasmid and cosmid DNA types, to name but a few.



  When an adenovirus is used as an expression vector, the LAV / HTLV III gene is ligated to an adenovirus transcription / translation control complex, for example the last promoter sequence and the tripartite leader sequences. This chimeric gene is then inserted into the genome of the adenovirus by recombination "in vitro" or "in vivo". Insertion into a non-essential region of the virus genome (for example the E1 or E3 region) will give a recombinant virus which is viable and capable of expressing, in infected hosts, the LAV / HTLV III related protein. Currently, there are two strains of adenovirus (types 4 and 7) approved and used as vaccines for military personnel. They are the first candidates to be used as vectors to express LAV / HTLV III genes.



  In addition, one can choose a host cell strain which modulates the expression of the inserted sequences, or modifies and processes the chemical gene produced in the specific manner desired. The expression of certain promoters can be increased in the presence of certain inducers (for example zinc and cadmium ions for metallothionein promoters). Therefore, we can regulate the expression of the LAV / HTLV III protein obtained by genetic engineering. This is important if the protein product of the cloned foreign gene is lethal to the host cells. In addition, modifications (eg glycosylation) and processing (eg cleavage) of the protein products are important for the functioning of the protein. Different host cells have characteristic and specific mechanisms for posttranslation processing and protein modification.

  Appropriate cell lines or host systems can be chosen to ensure correct modification and processing of the expressed foreign protein.



  In two particular embodiments described in detail in the examples included in the present invention, the sequences encoding the envelope of chimeric LAV / HTLV III were ligated, both in their complete form and in their portions, or sequences coding LAV / HTLV III gag on the 7.5K promoter of vaccinia virus to form chimeric genes in various plasmids. The chimeric genes of these plasmids were surrounded by additional vaccinia virus sequences, homologs of the vaccinia virus TK gene. The construction of the chimeric gene involved the use of nucleotides, both natural and synthetic, encoding control signals for the transcription and translation of the envelope sequences of LAV / HTLV III or the gag sequences of LAV / HTLV III.

  These chimeric genes were then introduced into the vaccinia virus expression vectors by "in vivo" recombination between the homologous TK region present on the plasmid vector and the vaccinia virus genome. These recombinant viruses containing the chimeric gene have been used as expression vectors to produce proteins related to the LAV / HTLV III envelope or to produce proteins related to LAV / HTLV III gag.



  In other particular embodiments detailed in the examples included in the present invention, the envelope coding sequences of LAV / HTLV III or the gag coding sequences of LAV / HTLV III were ligated to the promoter. polyhedrin of the Autoqrapha Californica nuclear polyhedrosis virus (AcNPV) to form chimeric genes in various plasmids. The chimeric genes in these plasmids were surrounded by additional sequences of AcNPV. The construction of chimeric genes involved the use of natural nucleotide coding control signals for the transcription and translation of LAV / HTLV III gag or envelope sequences. The chimeric genes were then introduced into the AcNPV expression vectors, by in vivo recombination between the homologous DNAs present on the plasmid vector and the AcNPV genome.

  These recombinant viruses containing the chimeric genes have been used as expression vectors to produce proteins related to the LAV / HTLV III envelope or gag related proteins of LAV / HTLV III.


 IDENTIFICATION OF RECOMBINANT EXPRESSION VECTORS CAPABLE OF REPLICATION AND EXPRESSION OF THE INSERTED GENE
 



  Expression vectors containing inserted sequences of foreign genes can be identified by three general approaches: (a) DNA-DNA hybridization; (b) presence or absence of "marker" gene functions, and (c) expression of inserted sequences. In the first approach, the presence of a foreign gene inserted into an expression vector can be detected by DNA-DNA hybridization, using probes comprising sequences which are homologous to the inserted foreign gene. In the second approach, we can identify and choose the recombinant vector / host system based on the presence or absence of certain functions of "marker" genes (for example thymidine kinase activity, resistance to antibiotics , a transformation phenotype, the formation of an occluded body in baculovirus) due to the insertion of foreign genes into the vector.

   For example, if the LAV / HTLV III gene is inserted into the vector marker gene sequence, the recombinants containing the inserted LAV / HTLV III sequence can be identified by the absence of the gene function. marker pen. In the third approach, recombinant expression vectors can be identified by looking for and / or assaying the product of the foreign gene expressed by the recombinant. Such research or assays may be based on the physical, immunological or functional properties of the gene product.



  Once a particular molecule of recombinant DNA has been identified and isolated, several methods can be used to cause its propagation, depending on whether or not such a recombinant constitutes a self-replicating unit (a replicon). A self-replicating unit, for example plasmids, viruses, cells, etc., can multiply in the appropriate cellular environment and under appropriate growth conditions. Recombinants lacking a self-replicating unit must be integrated into a molecule comprising such a unit for propagation to take place. For example, certain plasmid expression vectors must, when introduced into a host cell, be integrated into the cell chromosome to guarantee propagation and stable expression of the recombinant gene.

  Once a suitable host system and suitable growth conditions have been established, the recombinant expression vectors can be propagated and prepared in quantity.



  In particular embodiments included in the invention described in detail in the examples, chimeric genes containing the sequence encoding the envelope or gag of LAV / HTLV III have been inserted into the TK gene of the virus genome. vaccinia, thus transforming the virus into TK <->, that is, by destroying the ability of the virus to make thymidine kinase. Such recombinants were chosen on the basis of their ability to grow in media containing 5-bromo-deoxy-uridine, which is a nucleoside analogous to that fatal for TK cells. <+> but not for TK cells <->. Furthermore, recombinants were identified by DNA-DNA hybridization, using specific LAV / HTLV III envelope probes or specific LAV / HTLV III gag probes.

  The recombinant TK virus was isolated <-> by plaque purification and reserve stocks were prepared from infected tissue culture cells.



  In other particular forms of implementation included in the invention, described in detail in the examples, chimeric genes containing the envelope or gag coding sequence of LAV / HTLV III have been inserted into the polyhedrin gene of the genome of AcNPV, thereby transforming the virus into a non-occlusion phenotype. Such recombinants were chosen visually based on their absence of particles of occluded virus. The recombinants were also identified by Northern (North) coagulation analysis using specific LAV / HTLV III envelope probes or specific LAV / HTLV III gag type probes. Recombinant viruses were isolated by plaque purification and stock stocks were prepared from infected tissue culture cells.


 IDENTIFICATION AND PURIFICATION OF THE EXPRESS GENE PRODUCT
 



  Once a recombinant which expresses the LAV / HTLV III gene has been identified, the gene product should be analyzed. This can be achieved by analyzes and dosages based on the physical, immunological or functional properties of the product. An immunoassay is particularly important when the end goal is to use the products of recombinant genes or viruses that express such products in vaccine formulations and / or as antigens in immunoassays for diagnostics.



  Various antisera are available to analyze the immunoreactivity of the product, which includes, but is not limited to, serum from patients suffering from LAS (lymphadenopathy syndrome) or AIDS and polyvalent anti-sera directed against the LAV virus / HTLV, the viral envelope protein or core proteins encoded by the gag gene. Immunogenic molecules include analogs produced in bacterial systems, or synthetic peptides containing antigenic determinants of the LAV / HTLV III envelope or LAV / HTLV III nucleus antigens. The identification of the peptides and proteins described in the present invention is based on two requirements. First, the LAV / HTLV III envelope-related protein must be produced only in cells infected with recombinant virus.

  Secondly, the related protein LAV / HTLV III must be capable of an immunological reaction with the serum of patients suffering from AIDS or with various antibodies directed against the envelope or core proteins of LAV / HTLV III or their analogues and derivatives.



  The protein must be immunoreactive, whether it results from the expression of the entire gene sequence, part of a gene sequence, or two or more gene sequences that are ligated to direct the production of fusion proteins. This reactivity can be demonstrated by conventional immunological techniques, such as radioimmunoprecipitation, radioimmunological competition or immunocaillots.



  Once the protein related to LAV / HTLV III has been identified, this protein can be isolated and purified by conventional methods comprising chromatography (for example chromatography by ion exchange, by affinity and in a column of classification by dimensions), by centrifugation, by difference in solubility, or by any other conventional technique for the purification of proteins.



  Alternatively, once an immunoreactive protein related to LAV / HTLV III and produced by a recombinant has been identified, the amino acid sequence of the immunoreactive protein can be deduced from the nucleotide sequence of the chimeric gene contained in the recombinant. As a result, the protein can be synthesized by conventional chemical methods known in the art (see, for example, M. Hunkapiller et al., 1984, Nature 310: 105-111).



   In particular embodiments included in the present invention, such peptides, whether produced by techniques using recombinant DNA or by methods of chemical synthesis, include, without this list being limiting, all or part of the amino acid sequences essentially as shown in Figure 2 or Figure 14, which includes altered sequences in which functionally equivalent amino acid residues replace residues within the sequence, which creates a silent variation . For example, one or more amino acid residues in the sequence can be replaced by another amino acid of similar polarity, which acts as a functional equivalent, giving a silent alteration.

  One can choose, among other members of the class to which the amino acid belongs, elements to replace an amino acid within the sequence. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged amino acids (acids) include aspartic acid and glutamic acid.


 DETERMINATION OF THE IMMUNE POWER OF THE RECOMBINANT PRODUCT
 



  The immune potency of the LAV / HTLV III-related product can be determined by monitoring the immunological response of test animals after immunization by injection of the purified protein or purified synthetic peptide or protein. When the LAV / HTLV III related protein is expressed by an infectious recombinant virus, the recombinant virus itself can be used to immunize test animals. Test animals can include mice, rabbits, chimpanzees, and possibly humans can also be used. Methods of introducing the immunogen may include the intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal or any other conventional route of immunization.

  The immunological response of the test subjects can be analyzed by three approaches: (a) the reactivity of the resulting immune serum with respect to authentic viral antigens of LAV / HTLV III, according to assays or tests carried out by techniques known, for example the enzyme linked immunosorption assay (ELISA), immunocoagulates, radioimmunoprecipitations, etc. (b) the ability of the immune serum to neutralize the infectivity of LAV / HTLV III "in vitro" (M. Robert-Guroff, 1985, Nature 316: 72-74) (for detection of the anti-envelope antibody ), and (c) protection against infection by LAV / HTLV III and / or alleviation of infectious symptoms in immunized animals (DP Francis, 1984, Lancet 2: 1276-1277; DC Gujdusek, 1985, Lancet 1: 55-56).


 FORMULATION OF A VACCINE
 



  The aim is to formulate a vaccine in which the immunogen is related to an LAV / HTLV III epitope or which contains a recombinant virus which expresses such an immunogen which protects against infections with LAV / HTLV III virus for the prevention of lymphadenopathy (LAS) or AIDS. In addition, multivalent vaccine formulations can be prepared which contain more than one immunogenic determinant related to LAV / HTLV III. Such vaccines include, but are not limited to, those containing LAV / HTLV III envelope related epitopes formulated in isolation or in combination with other LAV / HTLV III epitopes, such as gag related epitopes. Such multivalent vaccines, containing both envelope coded and gag coded epitopes, may be important for preventing the development of AIDS.

  People without symptoms seem to make anti-gag antibodies more often than sick patients, while the two groups of patients make antibodies against envelope determinants (Science 1986, 233: 419). Studies have also shown that AIDS patients make antibodies to the envelope and nucleus (gag) proteins in the early stages without symptoms. As the disease progresses and symptoms appear, anti-gag antibodies are reduced, while anti-envelope antibodies remain (Science, 1986, 233-282).

  Thus, the inclusion of gag-related epitopes in vaccine formulations, the production of which is described as a specific aspect of carrying out the present invention, may be important for immunoprophylaxis or immunotherapy in the case LAV / HTLV III-related disease.



  Multivalent vaccines can be formulated to contain the LAV / HTLV III gene products and / or recombinant viruses expressing the chimeric gene products. They can also be used in combination with other immunogens for the prevention of lymphadenopathy syndrome (LAS) or AIDS and other diseases. Examples of various formulations are presented below.


 VIRUS VACCINE FORMULATIONS
 



  A live recombinant virus vaccine or an inactivated recombinant virus vaccine can be formulated. The choice depends on the nature of the recombinant virus that is used to express the epitopes related to LAV / HTLV III. When the recombinant virus is infectious for the host to be immunized but does not cause disease, a live vaccine is preferable because multiplication in the host leads to prolonged stimulation of a kind and amplitude similar to that which occurs in natural subclinical infections and, therefore, it confers a strong immunity of long duration. When introduced into a host animal, the infectious recombinant virus can express proteins related to LAV / HTLV III from its chimeric gene and thus stimulate an immunological response against antigens of LAV / HTLV III.

   When such an immunological response provides protection against further attack by LAV / HTLV III, the live recombinant virus can itself serve as a preventive vaccine against infection by the AIDS virus. The production of such a recombinant virus for use in these formulations can involve both "in vitro" (eg, tissue culture cells) and "in vivo" systems (eg, a natural host animal such as a cow). Conventional methods for the preparation and formulation of smallpox vaccines can be adapted to the formulation of a recombinant virus vaccine (for example, see Vaccinia Viruses and Factors for Vaccine Antigens, published under the direction of GU Quinnan at Elsevier, 1985, pages 109-116).



  Multivalent live virus vaccines can be prepared from a single or small number of infectious recombinant viruses that express multiple epitopes related to LAV / HTLV III / HTLV. The vaccine may also include viruses that express epitopes from organisms causing other diseases, in addition to the LAV / HTLV III epitopes. For example, a vaccinia virus (which can receive approximately 35 kp base of foreign DNA) can be genetically engineered to contain coding sequences of LAV / HTLV III epitopes related to the envelope and to gag. The virus can also encode other epitopes in addition to those concerning LAV / HTLV III. Such a recombinant virus itself can serve as an immunogen in a multivalent vaccine.

  Alternatively, a mixture of vaccinia or other viruses, each capable of directing the expression of a different gene encoding different epitopes of LAV / HTLV III and / or other organisms capable of causing disease.



  Whether or not the recombinant virus is infectious to the host to be immunized, an inactivated vaccine formulation can be prepared. Inactivated vaccines are "dead" in the sense that their infectivity has been destroyed, usually by treatment with formaldehyde. In principle, the infectivity of the virus is destroyed without affecting the proteins of the capsid or envelope which carry the immunogenic character of the virus. In order to prepare inactivated vaccines, large amounts of recombinant virus must be grown in culture to provide the necessary amount of related antigens. A mixture of inactivated viruses that express different epitopes can be used to formulate "multivalent" vaccines.

  In some cases, this may be preferable to live vaccine formulations, due to potential difficulties due to mutual interference of live viruses administered together. In either case, the inactivated recombinant virus, or a mixture of such viruses, should be formulated with a suitable adjuvant to enhance the immunological response to their antigens. Suitable adjuvants include, but are not limited to, mineral gels, for example aluminum hydroxide; surfactants such as lysolecithin, "Pluronic" polyols; polyanions; peptides; emulsions in oil and adjuvants potentially useful for human beings, such as BCG (bacillus Calmette Guerin) and corynebacterium parvum.



  Many methods can be used to introduce the vaccine formulations described above; they include, without being limiting, the intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous and intranasal routes. When using a formulation of live recombinant virus vaccines, this vaccine can be introduced by the natural route of infection of the related wild type virus which was used to obtain the recombinant virus in the vaccine formulation.


 FORMULATION OF SUB-UNIT VACCINES
 



  Instead of virus-based vaccines, the LAV / HTLV III-related protein itself can be used as an immunogen in subunit vaccine formulations which may be multivalent. As previously explained, subunit vaccines include only the involved immunogenic material necessary to immunize a host. Therefore, the LAV / HTLV III related protein (s) can or can be purified from recombinants which express the LAV / HTLV III epitopes.

  Such recombinants include any of the cultured cells infected with the virus, bacterial transformants, yeast transformants or insects infected with viruses which express the epitopes of LAV / HTLV III (reference may be made to what has been said above about the insertion of protein coding sequences, the identification of recombinant expression vectors, and about the identification and purification of the expressed gene product). In another embodiment of the present invention, the LAV / HTLV III related peptides or proteins can be obtained by chemical synthesis.

  For this, the amino acid sequence of such a peptide or protein can be deduced from the nucleotide sequence of the chimeric gene which directs its expression (see, for example, what has been indicated below). above regarding identification and purification of the expressed gene product).



  Whether the immunogens are purified from recombinants or obtained by chemical synthesis, the final product can be adjusted to an appropriate concentration and formulated with any suitable vaccine adjuvant and then packaged for use. Suitable adjuvants include, but are not limited to, mineral gels, for example aluminum hydroxide, surfactants such as lysolecithin, "Pluronic" polyols; polyanions; peptides; emulsions in oil and adjuvants potentially useful for human beings, such as BCG (Bacille Calmette Guerin) and corynebacterium parvum. The immunogen can also be incorporated into liposomes, or conjugated with polysaccharides and / or other polymers for use in a vaccine formulation.



   When the LAV / HTLV III related peptide or protein is a hapten, i.e. a molecule which is antigenic in that it can react selectively with recognized antibodies, but which is not antigenic in that it that it cannot trigger an immune response, the hapten can be covalently linked to an immunogenic support or molecule; for example, a large protein such as HIND-3 introduced into Pv-env5 and the EcoRI site filled in plasmid P26 created a stop codon in phase with the coding sequence for the envelope. This intermediate construction was called Pv-env5 / 26.



  The plasmid pv-env5 / 26 DNA was used to transform, amplify and purify E.coli MC1000. It was then subjected to digestion with BamHI and HpAI, and the resulting fragments were resolved on a gel agar at 1%. The 2.1 kilopair base fragment containing the LAV / HTLV envelope coding sequence was isolated, and then ligated to plasmid pGS 20 previously digested with BamHI and SmAI . The resulting plasmid Pv-env 7 contains the 7.5 K vaccinia virus promoter ligated to a specific sequence of LAV / HTLV III from the 96 base pairs upstream of the relative priming codon at the HIND 3 site at nucleotide number 6798 (Figure 5).

  This plasmid therefore does not contain the presumed sequence for anchoring the LAV / HTLV III envelope gene.


 CONSTRUCTION AND CHARACTERIZATION OF THE RECOMBINANT VACCINE VIRUS CONTAINING THE CHIMERIC GENE OF LAV / HTLV III ENVELOPE
 



  Two starting strains of vaccinia virus were used for the construction of the recombinant viruses: WR, the research control strain, and v-NY, a derivative of the strain originating from the "New-York City Board of Health" (Service de New York City Public Health).



  The insertion of the chimeric gene sequences corresponding to the LAV / HTLV III envelope into the genome of the vaccinia virus is obtained by "in vivo" recombination, made possible by the fact that the chimeric genes are surrounded in the pv plasmids -env2, pv-env5 and pv-env7 by vaccinia virus sequences encoding the thymidine kinase (TK) gene. The introduction of these plasmids into cells infected with the vaccinia virus allowed recombination between the TK sequence of the plasmid and the homologous sequence of the vaccinia virus genome. The insertion of the chimeric gene occurs as a result of double recombination in the lateral entourage sequences. Such recombinants will contain the chimeric gene inserted into the TK gene of vaccinia and, therefore, their phenotype will correspond to TK <->.

  We can choose these TK recombinants <-> to make them grow in a medium to which we added BUdR, which is lethal for TK cells <+> but not for TK cells <->. The general principle of this procedure has been described (M. Mackett, G.L. Smith and B. Moss, 1984, J. Virol. 49, 857-864).


 CONSTRUCTION OF THE RECOMBINANT VACCINE VIRUS CONTAINING THE CHIMERIC GENE CORRESPONDING TO THE LAV / HTLV III ENVELOPE
 



  A 100 mm diameter dish containing 80% confluent African green monkey kidney cells (strain BSC-40) was infected with vaccinia virus (strain WR) at a multiplicity of infections of 0.05 . After 2 to 4 hours of incubation at 37 DEG C, the infected cells were covered with coprecipitates, using calcium phosphate, the plasmid DNA pv-env2 or pv-env5. The precipitates were prepared by adding 0.5 ml of a 2XADN-CaCl2 solution dropwise to 0.5 ml of 2XHeBS solution (the 2XADN-CaCl2 solution contains 20 μg of plasmid DNA in 0.5 ml 0.25M CaCl2; 2XHeBS contains, per ml, 16 mg NaCl, 0.74 mg KCl, 0.25 mg Na2HPO4 2H2O, 2 mg dextrose, and 10 mg HEPES, at pH 7.08 ). The coprecipitate obtained using DNA-calcium phosphate was allowed to form at room temperature for 30 minutes.

  Four hours after covering them with precipitate, the cells were washed once with 1XHeBs, incubated at 37 DEG C for 3 minutes in the presence of 2 ml of 15% glycerol in 1XHeBs, then washed once more with 10 ml of growth medium (MEMD + 10% fetal calf serum + 100 units / ml of penicillin and 100 units / ml of streptomycin). Two days later, the infected cells were harvested and collected by centrifugation (4 DEG C, 10 minutes at 2000 x g). Virus stocks were prepared by resuspending these cells in 1 ml of phosphate buffered saline with added bovine serum albumin, followed by two freeze-thaw cycles and three treatments. for 15 seconds by ultrasound.



  We chose recombinants by placing 0.1 ml of dilution at 10 <-> <3> virus stocks from the above operations on circular plates in 60 mm dishes of confluent human cells 143 TK <->, topped with 5 ml / box of 1% Nobel agar (Difco, Detroit, Michigan) with 5% calf serum, 25 mu g / ml of BUdR in MEMD. Two days later, cells were stained by placing 2 ml / box of agar medium containing the same ingredients as above, plus 0.01% neutral red.

  The individual plates were removed one day after staining, resuspended in 0.5 ml of physiological phosphate buffered saline supplemented with bovine serum albumin (PBSAM), and used aliquots (0.25 ml) of virus suspensions to infect confluent 143 TK cells <-> placed in 16 mm diameter pits under a selective medium (MEMD + 10% fetal calf serum + 100 units / ml streptomycin + 100 units / ml penicillin + 25 mu g / ml BUdR) . The infected cells were collected by centrifugation and resuspended in 100 μl of physiological phosphate buffered saline (PBS) containing 0.5 mg / ml of trypsin and 0.2 mg / ml of EDTA (acid ethylene diamine tetra acetic).

   The cells were lysed by incubation at 37 DEG C for 30 minutes, which was followed by 3 cycles of 20 seconds each of ultrasound treatment. Cell lysates were collected on nitrocellulose filters, using a manifold or multi-pit filtration manifold (Schleicher and Schuell, Arlington, MA). DNA-DNA hybridization, as described (M. Mackett, GL Smith and B. Moss, 1982; Proc. Nat'l. Acad. Sci. 79, 7415-7419), was determined to determine the presence of the DNA sequences specific for the LAV / HTLV III envelope in these samples. DNA labeled plasmid pRS-3 was used as the hybridization probe. <3> <2> p, prepared by appropriate translation.

  Recombinant recombinants which gave positive hybridization for this probe were further purified twice on 143 TK cells. <-> under selective conditions (medium containing BUdR) and once on BSC-40 cells under non-selective conditions. After final confirmation by DNA-DNA hybridization, virus stocks were prepared from the three times purified plates on BSC-40 cells and used for subsequent characterization.



  A schematic representation of the construction of the recombinant viruses is shown in FIG. 6, in which the gene sequence corresponding to the envelope of LAV / HTLV III (env) located downstream of the 7.5 K vaccinia promoter (p-> ) is surrounded within the vaccinia genome by TK DNA sequences. The stocks of virus originating from the recombination between the genome of vaccinia virus and the plasmid pv-env5 were called v-env5, and they contain the entire gene of the envelope of LAV / HTLV III. In the particular embodiment described in the examples here, v-env5 contains the entire envelope gene as well as 96 base pairs of the untranslated sequences close to 5 min and 223 base pairs of the untranslated sequences close 3 min.

  The virus stocks from pv-env2 have been called v-env2 and they contain most of the LAV / HTLV III envelope gene (i.e., the KpnI fragment), but they do not contain the part of the sequence encoding the first 42 amino acids of the LAV / HTLV III envelope protein. Since the supposed signal sequence of the LAV / HTLV III coat protein is located within the first 49 amino acids of the protein, v-env2 should produce a protein that does not have the signal sequence; therefore, it should be expected that the LAV / HTLV III-related protein produced by this recombinant virus will not be transported to the membrane. In contrast, v-env5, which contains the entire LAV / HTLV III envelope sequence, must produce a protein that is transported to the membrane.

  Stocks of viruses originating from pv-env7 are called v-env7 and contain the complete nitrogen termination of the gene corresponding to the LAV / HTLV III envelope, but do not contain the sequence downstream of the HIND III site. Since the presumed transmembrane (anchor) sequence is missing in this construct, one would expect that the LAV / HTLV III-related protein produced by this recombinant will be secreted into the growth medium. This property is advantageous for the purification of this protein for use as a diagnostic reagent or for its formulation as a subunit vaccine.


 FORMATION OF RESTRICTION FRAGMENTS OF LAV / HTLV III VACCINE-ENVELOPE RECOMBINANTS
 



  As previously described (Esposito et al, 1981, J. Virol. Methods 2.2. 1975-180), the strain DNA of the starting vaccine viruses WR and v-NY, as well as their derivatives v-env5 and v-env5NY, respectively. They were digested with the restriction enzyme HIND III and the resulting fragments were resolved on a 0.7% agar gel. DNA from the starting strain WR presented the typical restriction fragment model (Figure 2A) as previously described (De Filippes, 1982, J. Virol. 43: 136-149). The restriction fragment model for v-NY was similar, but distinct from that obtained with WR. The most obvious differences were in the length of fragments b and c obtained with HIND III (HIND III fragments b and c), which are terminal fragments of the genome of the vaccinia virus.

  The v-env5 and v-env5NY recombinants do not contain HIND III J fragments of their starting or parental strains. Two new fragments were observed instead, having a size of 5.4 Kbp (kilopairs of bases) and 3.2 Kbp. This observation agrees with the result of the double recombination which inserted the sequence encoding the envelope of LAV / HTLV III into the TK gene of vaccinia, which is located on the HIND III J fragment. Two fragments were generated by the insertion , due to the presence of a HIND III site in the LAV / HTLV III sequence. This result also confirms the orientation of the LAV / HTLV III inserted fragment with respect to the vaccinia virus TK gene.


 SOUTHERN-TYPE COAGULATION ANALYSIS (SOUTH) OF LAV / HTLV III VACCINE-ENVELOPE RECOMBINANTS
 



  The identity of two new HIND III fragments in the restriction digests of recombinant viral genomes was confirmed by Southern type coagulation analysis. DNA fragments, resolved on an agar gel like that presented in FIG. 7A, were transferred to nitrocellulose filters, and were hybridized on a probe obtained by specific translation of the LAV / HTLV III envelope sequences. . As shown in Figure 7B, hybridization was detected only in the 5.4 and 3.2 Kbp fragments. This result confirms the structure of the genome of the recombinant virus as shown in FIG. 6.


 EXPRESSION OF LAV / HTLV III-LIKE PROTEINS IN TISSUE CULTURE CELLS INFECTED WITH RECOMBINANT VACCINE VIRUSES
 



  Recombinant vaccinia viruses have been shown to carry the LAV / HTLV III envelope chimeric genes capable of expressing proteins related to the LAV / HTLV III envelope during infection of cells in a culture. tissue. These proteins have also been found to be immunoreactive with serum from AIDS patients.


 IDENTIFICATION, USING IMMUNOCOAGULATION TECHNIQUES, OF PROTEINS RELATED TO THE LAV / HTLV III ENVELOPE EXPRESSED IN CELLS INFECTED WITH RECOMBINANT VACCINE VIRUS
 



   A 100 mm circular dish plate was infected with BSC40 confluent cells, at an infection rate or multiplicity of 10, with wild-type vaccinia virus or with its recombinant derivatives, v-env5 or v-env2. The infection was allowed to continue for 12 hours, after which cells were harvested, washed once with physiological phosphate buffered saline (PBS) and collected by centrifugation. The pellets of infected cells were resuspended in 1 ml of Laemmli sample buffer (Laemmli, U.K. 1970, Nature 227: 680) and lysis was carried out by 4 minutes of boiling. The total cellular protein in an aliquot (75 μl) of cell lysate was resolved by electrophoresis, on an SDS-polyacrylamide gel at a gradient of 7 to 15%.

  A purified LAV virion sample and an aliquot of lysate from cells "infected" with a non-pathogenic virus (ie, uninfected) were included as a control. The contents of the gel were passed through electrotransfer on a nitrocellulose filter sheet. The filter was incubated first in 5 ml of PBS (physiological saline solution buffered by phosphates) plus 5% defatted milk powder, for 20 minutes at room temperature and then for 2 hours at room temperature in PBS + 5% defatted milk powder + human serum from AIDS patients (1: 100 dilution of inactivated serum by heating). The filter was then washed five times with PBS + 0.05% "Tween 20" (polyoxyethylene sorbitan monolaurate) and once with PBS alone.

  The filtrate thus washed was incubated for 2 hours at room temperature with 5 ml of PBS containing 1% normal goat serum (inactivated by heating) plus a 1/3000 dilution of anti-human IgG conjugate (immunoglobulin G) goat cheese / horseradish peroxidase. The same filter was washed again 5 times with PBS + 0.05% "Tween 20", and once with TBS (0.5 M NaCl + 20 mM Tris-HCl, pH 7.5). The horseradish peroxidase conjugate attached to the filter was detected by reacting staining reagents, such as chloronaphthol, with the filter for 10 minutes at room temperature in the dark (the staining reagent such as chloronaphthol was prepared by mixing solutions A and B just before use Solution A: 20 ml of cold 30% methanol + 60 mg of 4-chloro-1-naphthol; solution B: 60 mu l of cold 30% hydrogen peroxide + 100 ml of TBS).

  Proteins attached to the filter and which have reacted with antibodies in the AIDS patient's serum can be detected by the staining reagent, by binding to the anti-human goat IgG / horseradish peroxidase conjugates.



  The results of this analysis, as shown in Figure 8A, indicated that recombinant vaccinia virus v-env5 produced a family of three proteins which were specifically immunoreactive with serum from an AIDS patient. These proteins had electrophoretic mobilities similar to those of the glycoproteins gp150, gp110 and gp41 of the authentic LAV / HTLV III virus, which are thought to be coded by the envelope gene (W. Robey et al., 1985, Science 228: 593-595). These proteins were not produced in cells infected with non-infectious virus or in cells infected with wild-type vaccinia virus.

  Recombinant V-env, which does not contain the neighboring sequences of 5 min coding for the supposed initiation methionine and the first 42 amino acids of the LAV / HTLV III envelope proteins, produced a protein of limited dimensions, but still immunoreactive in respect of a serum from an AIDS patient. It is assumed that the translation of the env sequence into recombinant v-env2 starts from the codon AUG transcribed from the sequence corresponding to the binding agent used in the construction of this recombinant (see above the construction of the plasmid vectors containing vaccinia virus ligated to the LAV / HTLV III envelope gene sequences encoding in 3 min).



  In a second experiment, two cell lines (BSC-40 and HeLa) were infected with v-env5 and v-env2. The specific LAV proteins were subjected, in lysates of infected cells, to assay tests by Western type immunocoagulation, as described below.



  Confluent monolayers of BSC-40 or HeLa cells were infected with v-env5 or v-env2 of recombinant viruses at a multiplicity or infection rate of 50 plaque-forming units per cell. Twelve hours after infection, the cells were washed twice in phosphate buffered saline, resuspended in Laemmli sample buffer and boiled for 5 minutes. Proteins from infected cells were resolved by polyacrylamide gel electrophoresis using sodium dodecyl sulfate (SDS-PAGE), subjected to electrotransfer to a nitrocellulosic membrane and reacted with pooled serum from people with positive serum in the search for LAV / HTLV-III.

  Immunoreactive proteins were detected using protein A, which was labeled with 125 I (Amersham, MA) by the chloramine T method. In Figure 8B, lanes 1 and 5 contain proteins from cells "infected" with non-infectious viruses; lanes 2 and 6 contain proteins from cells infected with wild-type vaccinia virus; lanes 3 and 7 of proteins from cells infected with v-env-5; and lanes 4 and 8 are from cells infected with v-env2. As a control (LAV / HTLV III), virion proteins of LAV / HTLV III purified using a sucrose gradient were used, and the positions of the envelope proteins gp150, gp110 and gp41 were as indicated on the figure. Molecular weights were expressed in kilodaltons (kd).



  Three major proteins capable of an immunological reaction ("immunoreactive") have been detected in v-env5 infected cells (FIG. 8B, lanes 3 and 7) with pooled serum from serum people giving a positive response ("seropositive"). "). The molecular weights of these proteins were estimated to be 150 kd, 120 kd and 41kd, similar to those of the LAV / HTLV III envelope glycoproteins gp150, gp110 and gp41.



   Recombinant virus V-env2 did not contain the sequence expected to signal for the LAV / HTLV III envelope, but could produce at least three immunoreactive polypeptides having molecular weights 99 kd, 68 kd and 40 kd (Figure 8B, lanes 4 and 8).


 IDENTIFICATION, BY IMMUNOPRECIPITATION TECHNIQUES, OF PROTEINS RELATED TO THE LAV / HTLV III ENVELOPE AND EXPRESSED IN CELLS INFECTED WITH RECOMBINANT VACCINE VIRUS
 



  The immunoprecipitation assay, described below, showed that cells infected with recombinant vaccinia viruses of the present invention synthesize specifically immunoreactive proteins with serum from AIDS patients.



  A 100 mm dish containing BSC-40 confluent cells was infected with wild-type vaccinia virus, or its v-env5 or v-env2 recombinants, at an infection rate or multiple of 10. After a time interval of 9.5 hours after infection, the growth medium was replaced with MEMD without methionine and without additional serum. At 10 hours after infection, the medium was replaced by 2 ml of MEMD without methionine containing 100 μm Ci / ml of ( <3> <5> S) methionine and the labeling was allowed to continue for 2 hours at 37 DEG C. At the end of the labeling period, the cells were washed once with physiological phosphate-buffered saline (PBS) ) and collected by centrifugation.

  The cell pellets were resuspended in 1 ml of lysis buffer: 1% NP 40 (polyoxyethylene (9) p-tertoctylphenol), 0.5% sodium deoxychlolate, 0.1M NaCl, tris-HCl 0 , 01M, pH 7.4, 1 mM EDTA, and the lysate was separated by centrifugation for 1 minute in an Eppendorf microcentrifuge.



  Immunoprecipitation was performed by adding 5 microliters of heat-inactivated human serum from a control population or from AIDS patients to 100 µl of cell lysate. After 1 hour of incubation at 4 DEG C, 60 μl of activated Staphylococcus aureus cells ("Pansorbin" cells, Calbiochem-Behring Corp., LaJolla, CA) were added and the incubation was allowed to continue for 1 additional hour at 4 DEG C. The immunoprecipitation complexes were collected by centrifugation for 30 seconds in an Eppendorf microcentrifuge at 4 DEG C and washed once in 1M NaCl + 0.1% NP 40 + Tris -HCl 0.01 M, pH 7.4, and twice RIPA buffer (10 mM Tris-HCl, pH 7.2, 0.15 M NaCl, 1% sodium deoxycholate, 1% "Triton- X 100 "(polyoxyethylene (9-10) p-tert-octyl-phenol), 0.1% sodium lauryl sulfate).

  The immunoprecipitates were resuspended, washed in 50 μl of Laemmli sample buffer, boiled for 1 minute and centrifuged for 1 minute in an Eppendorf microcentrifuge. The immunoprecipitated proteins present in the supernatant were analyzed by electrophoresis on polyacrylamide gels containing 15% SDS. After electrophoresis, the gels were stained with Coomassie blue dye, treated with sodium salicylate (30 minutes at room temperature in 1M sodium salicylate) and dried for fluorography.



  The results of this analysis, as shown in Figure 9A, indicated that recombinant v-env5 synthesized a family of proteins with specific immunoreaction with serum from an AIDS patient. These proteins had apparent molecular weights of 160, 140, 120, 42 and 40 kilodaltons (kd), corresponding approximately to the apparent molecular weights of the envelope-related glycoproteins cited for LAV / HTLV III (WG Robey et al., 1985, Science 228: 593-595). These proteins were not present in cells "infected" with non-infectious viruses or in cells infected with wild-type vaccinia virus, nor did they undergo immunoprecipitation with control human serum.

  In cells infected with v-env2, a truncated protein, with an apparent molecular weight of 95 kd, was produced and recognized by serum from an AIDS patient. This truncated form of protein related to the envelope of LAV / HTLV III most likely starts from the codon AUG coded by the sequence of binding agents located in 5 min near the inserted sequence, corresponding to LAV / HTLV III in this recombinant.


 MARKING BY <3> H-GLUCOSAMINE OF LAV / HTLV III ENVELOPE-LIKE PROTEINS PRODUCED BY RECOMBINANT VACCINE VIRUSES CORRESPONDING TO LAV / HTLV III
 



  The radioimmunoprecipitation assay described below indicated that the recombinant vaccinia viruses, corresponding to LAV / HTLV III of the present invention, produced glycosylated envelope proteins.



  HeLa cells were subjected to infection with non-infectious viruses (see Figure 9B, tracks 1 and 5) or to infection separately with wild-type vaccinia virus (tracks 1 and 6), recombinant v-env5 ( tracks 3 and 7) or v-env2 (tracks 4 and 8), all at an infection rate of 50 plaque-forming units per cell. Was added to the culture medium, between 4 and 16 hours after infection, glucosamine labeled <3> H (0.25 mu Ci at 23 mCi / mg, Amersham). Cells were washed twice with phosphate buffered physiological saline and lysed in buffer containing 0.1M NaCl, 0.01M tris-HCl pH 7.4, 1mM EDTA, 1% NP- 40 (polyoxyethylene (9) p-tert-octylphenol) and 0.5% sodium deoxycholate.

  Aliquots of cell lysate were mixed with normal human serum (lanes 1-4) or group serum from people with LAV / HTLV III positive serum (lanes 5-8). The immunoreactive proteins were precipitated by fixed cells of Staphyloccocus aureus carrying protein A, then resolution was carried out by SDS-PAGE and detection by fluorography.



  The results of labeling with glucosamine comprising a radioisotope, as represented in FIG. 9B, lane 7, indicate that the proteins obtained using the recombinant v-env5, such as the LAV / HTLV III envelope proteins have also been glycosylated. Differences in glycosylation configurations may explain the slight variations observed in the electrophoresis mobilities of proteins produced using recombinants, in comparison with the glycoproteins due to the LAV virion.



   As would be expected for proteins lacking a signal-emitting peptide, nitrogen-bound glycosylation was not observed, with <3> H-glucosamine, in cells infected with v-env2 (Figure 9B, lane 8).


 IMMUNOPRECIPITATION ANALYSIS, WITH HUNTING PULSIONS, OF PROTEINS RELATED TO THE LAV / HTLV III ENVELOPE PRODUCED BY RECOMBINANT VACCINE VIRUSES LINKED TO LAV / HTLV III
 



  It has been suggested that LAV / HTLV III gp150 is the precursor from which an external protein gp110 and a transmembrane protein gp41 originate. The "pulse-hunting" immunoprecipitation test described below indicates that the proteins at 150 kd, 120 kd and 41 kd obtained using the recombinant vaccinia virus corresponding to LAV / HTLV III have a relationship between precursor and product similar to that suggested for gp150, gp110 and gp41 for authentic LAV / HTLV III.



  Conjunct monolayers of HeLa cells were infected with wild-type vaccinia virus (see FIG. 9C, tracks 1-6), recombinant v-env (tracks 7-12) or v-env2 (tracks 13-18), all at a multiplicity of infection of 50 plaque-forming units / cell. After 10 and a half hours after infection, the cells were labeled with <3> <5> S-methionine (more than 800 Ci / mmol, Amersham) at 100 mu Ci / ml for 15 minutes. At the end of the markate period, the cells were washed once with 2 ml of preheated hunting medium (Eagle medium modified according to Dulbecco, MEMD, + 3 mg / ml of L-methionine + 5% calf serum + 100 units / ml of penicillin + 100 mu g / ml of streptomycin) then the cells were again supplied with 1 ml of the same medium before being returned to the incubator.

  At various times thereafter, the cells were washed and lyzed as described above above (labeling with <3> H-glycosamine) and the proteins from cell lysates were subjected to immunoprecipitation with pooled serum from people whose serum had a positive response for LAV / HTLV III. The immunoprecipitated proteins were resolved by electrophoresis on SDS-PAGE and detected by fluorography. The duration of each hunt was as follows: 0 hours (tracks 1, 7, 13); 1 half hour (tracks 3, 8, 14); 1 hour (tracks 3, 9, 15); 2 hours (tracks 4, 10, 16); 6 hours (tracks 5, 11, 17) and 12 hours (tracks 6, 12, 18).

  The results presented in FIG. 9C, tracks 7-12, indicate for the proteins at 150 kd, 120 kd and 41 kd obtained using the recombinant, the same precursor-product relationship as for gp150, gp110 and gp41 of LAV / HTLV III authentic. Processing of the 150 kd protein appeared slow and ineffective in HeLa cells, since after 6 hours after pulse labeling, less than 50% of the radioactivity of the 150 kd protein had passed into the proteins. 120 kd and 41 kd. Preliminary results indicate that this treatment is more effective in certain types of peripheral human blood cells infected with the same recombinant viruses.

  These experiments also indicated that the env sequence in v-env2, which did not include the sequence expected to signal to detect the LAV envelope, was expressed as an unmodified 87 kd precursor (780 amino acids) , which gave by treatment an intermediate at 99 kd and was cleaved into the polypeptides at 68 kd and 40 kd (Figure 9C, lanes 13-18).


 PRESENCE OF A LAV / HTLV III-LIKE PROTEIN IN THE GROWTH OF CELLS INFECTED WITH LAV / HTLV III VACCINE VIRUSES
 



  The radioimmunoprecipitation assay described below showed that the immunoreactive proteins produced by the recombinant vaccinia virus of the invention can be expressed and processed in a manner similar to the authentic LAV / HTLV III envelope glycoprotein.



  Conjugated monolayers of HeLa cells were subjected to "infection" with non-infectious virus (see FIG. 9D, lane 1) or separately to infection with wild-type vaccinia virus (lane 2), by recombinant v-env5 ( lane 3) or recombinant v-env2 (lane 4), all at a multiplicity or infection rate of 20 plaque-forming units per cell. The cells were labeled with <3> <5> S-methionine (more than 800 Ci / mmol, Amersham) at 100 mu Ci / ml 10 to 12 hours after infection. At the end of the labeling period, the medium was removed and clarified by centrifugation for 2 minutes at 12,000 g before being used for immunoprecipitation with pooled serum from people with positive results for LAV / HTLV III.

  The proteins originating from infected cells having undergone immunoprecipitation by the same serum are presented in the part marked "pellet" and the proteins coming from the growth or culture medium are presented in the part marked "Supe" (supernatant upper medium).



  As expected for LAV / HTLV III gp110, the 120 kd protein obtained using the recombinant was also detected in the infected medium (Figure 9D, lane 3). These results showed that the recombinant virus v-env5 was able to express the gene corresponding to the LAV envelope and to produce immunoreactive proteins which were treated in a similar manner to authentic LAV / HTLV III envelope glycoproteins. .



  As would be expected for proteins without signal peptides, polypeptides linked to the LAV / HTLV III envelope were not detected in the growth medium of cells infected with v-env2 (Figure 9D, track 4).



  In addition, cells infected with v-env7 expressed a truncated protein of 130 Kd. This protein does not contain the 217 terminal carbon amino acids containing the anchor sequences of the LAV / HTLV III envelope protein. This truncated protein (gp130) was secreted into the growth medium much more efficiently than gp110 or its precursor gp150 (Figure 9E). At the same time, gp130 is not efficiently transformed into gp110, although the cleavage site is still present on the truncated molecule.


 IMMUNITY OF THE RECOMBINANT VIRUS CORRESPONDING TO THE LAV ENVELOPE
 



   Recombinant viruses, carrying the chimeric gene corresponding to the envelope of LAV / HTLV III, have been shown to be able to trigger an antibody response against LAV / HTLV III in two strains of mice and a species of subhuman primate. .


 IMMUNOGENICITY OF VACCINE RECOMBINANTS CARRYING THE LAV / HTLV III ENVELOPE GENE IN MICE
 



  The immunogenicity of proteins expressed by v-env5 and v-env2 of recombinant vaccinia viruses was examined. The experiments described below indicate the ability of these recombinant viruses to trigger an immunological response for all the major LAV / HTLV III glycoproteins.



  Two mouse lines, one inbred (C57B16J) and the other non-inbred (ICR), were inoculated with v-env2 or v-env5 of recombinant viruses. All animals were 5 to 7 weeks old at the time of inoculation. Four routes of inoculation were used: leg pad, tail scarification, intranasal and intraperitoneal routes. The inoculation was carried out in the paw pad by injecting 25 mu l (5 x 10 <6> plaque forming units) of recombinant virus in each of the hind leg pads. The tail scarification was carried out by scraping, using a two-pointed needle, the skin at the base of the tail and applying to the scarified surface 10 mu l (2 x 10 <7> plaque forming units) of recombinant virus.

  An intranasal inoculation was carried out by placing 10 ml (2 x 10 <7> plaque forming units) of recombinant virus on the nose of the mice and letting the mouse aspirate the inoculum. The intraperitoneal injections were carried out by injecting 10 μl (2 × 10 <7> plaque forming units) of recombinant viruses in the peritoneal cavity of mice. Stock preparation and virus dilution were carried out by operating as described above with respect to cells and viruses.

  Serum samples were collected at 2 week intervals after inoculation of individual mice and these samples were analyzed as well by an enzyme linked immunosorption assay ("ELISA"; enzyme linked immunosorbent assay) only by Western-type coagulate analysis as described below.


 SERUM CONVERSION (SEROCONVERSION), SHOWN BY DOSAGE OR ELISA TEST, OF MICE IMMUNIZED BY RECOMBINANT LAV / HTLV III VACCINE VIRUSES
 



  The results of the ELISA tests on 6-week serum samples are summarized in Table 1. The v-env2 and v-env5 sequences of recombinant viruses converted (transformed) 100% and 95% the serum of the C57B16J mice, respectively. . For ICR mice, the serum conversion rate was 44% for v-env2 and 100% for v-env5.

  A total conversion of serum and the highest mean titer were obtained, in the ELISA test, in mice immunized by tail scarification.
 <tb> <TABLE> Columns = 4
 <tb> Title: Table I
 <tb> Head Col 01 to 04 AL = L: Seroconversion of mice inoculated with recombinant viruses carrying the chimeric gene of the LAV / HTLV III envelope
 <tb> SubHead Col 01 AL = L> Recombinant virus:
 <tb> SubHead Col 02 AL = L> Inoculation route:
 <tb> SubHead Col 03 to 04 AL = L:

  Seroconversion of inoculated mice *
 <tb> SubHead Col 03 AL = L> ICR:
 <tb> SubHead Col 04 AL = L> C57B16J:
 <tb> <SEP> v-env2 <SEP> leg pad <SEP> 1/3 <SEP> 4/5
 <tb> <SEP> tail scarification <SEP> 3/3 <SEP> 4/5
 <tb> <SEP> intranasal route <SEP> 0/3 <SEP> 4/5
 <tb> <SEP> intraperitoneal route <SEP> not performed <SEP> 3/5
 <tb> <SEP> v-env5 <SEP> leg pad <SEP> 4/4 <SEP> 5/5
 <tb> <SEP> tail scarification <SEP> 3/3 <SEP> 5/5
 <tb> <SEP> intranasal route <SEP> 3/3 <SEP> 4/5
 <tb> <SEP> intraperitoneal route <SEP> not performed <SEP> 5/5
 * Seroconversion, determined by the ELISA test as described, is expressed by the number of mice having seroconversion relative to the total number of mice having undergone inoculation, for each group.
  
 <tb> </TABLE>



  The results of the ELISA assays or tests were obtained as follows: inactivated and purified LAV / HTLV III virions were diluted in carbonate buffer (50 nM sodium carbonate, pH 9.6) and placed these dilutions in microtiter plates comprising 96 pits or wells (100 μl / hollow, containing 0.2 μg of inactivated LAV / HTLV III). The bond was allowed to proceed at 4 DEG C overnight. The unbound protein was aspirated and washed with 200 ml (by trough) of physiological saline with phosphate buffer + 5% defatted milk powder and then with 300 μl (by trough) at 4% of sucrose. After sucking up the excess sucrose solution, the plates were allowed to dry at room temperature (1 hour).

  Then 50 μl of physiological saline with phosphate buffer containing 2.5 μl of mouse serum sample were added to each well, and the reaction was allowed to proceed at 37 DEG C for 1 hour. At the end of the incubation period, the troughs were washed five times with physiological saline containing phosphate buffer (PBS) + 0.05% "Tween 20" (polyoxyethylene sorbitan monolaurate). 50 μl of goat anti-mouse immunoglobulin G / horseradish peroxidase conjugates were added to each trough (dilution 1: 5000 in PBS (physiological saline solution with phosphate buffer) + 0.05% of "Tween-20" , and allowed to react at 37 ° C. for 45 minutes After 5 washes with PBS + 0.05% of "Tween 20", 100 μl of a citrate-O-phenylenediamine-peroxide substrate were added.

  The substrate was prepared as follows: 0.294 g of sodium citrate dihydrate + 0.537 g of dibasic sodium phosphate crystal was dissolved in 10 ml of H2O and adjusted to pH 5.0 with HCl; 2 μl of 30% H 2 O 2 and 4 mg of O-phenylene diamine were added just before use. The plates were incubated for 30 minutes in the dark at room temperature. The reaction was stopped by adding 100 μl of 1.3N sulfuric acid to each trough and the optical absorption coefficient of the reaction mixture was measured at 490 nm.



  The results presented in table I are expressed in the form of the ratios of the total number of mice seropositive to the total number of mice having undergone an inoculation. Serum samples were collected 6 weeks after inoculation and analyzed by the ELISA assay assay. Serum samples from 5 uninoculated mice were used as a control. The mean ELISA titers for the control group were 0.021 +/- 0.005 and 0.017 +/- 0.010 for ICR and C57B15J mice, respectively. Serum samples from immunized inoculation mice with ELISA titers more than three standard deviations higher than those in the control group were considered positive.



   To show that mice inoculated with recombinant viruses ("seroconverted" mice) produced antibodies against authentic LAV / HTLV III envelope glycoproteins, serum samples from mice inoculated were analyzed ( "immunized mice") by a Western type immunocoagulation technique as follows: inbred male mice aged 5 to 7 weeks (C57B16J, Jackson Laboratory) were "immunized" by tail scarification, which consisted in apply a 10 mu l inoculum containing 2 x 10 <7> plaque-forming units of vaccinia virus recombinant to abrasions caused by scraping using a twin-pointed needle at the base of the tail. Animals were bled from the retro-orbital sinus 8 weeks after inoculation and the serum was kept frozen until needed.

  The aliquots of serum samples, diluted 50 times in a phosphate buffered saline + 0.2% "NP-40" and 3% non-fat milk powder, were reacted with virion proteins of LAV / HTLV III which were resolved by electrophoresis on polyacrylamide-SDS and immobilized by electrotransfer on nitrocellulose filters. The LAV / HTLV III proteins recognized by these sera were detected by goat anti-mouse immunoglobulin conjugated with alkaline phosphatases.



  The results obtained on these 8-week serum samples from C57B16J mice which have been inoculated with tail scarification of recombinant viruses are presented in FIG. 10. All the animals which have been inoculated with recombinant viruses have produced antibodies. who reacted with the LAV envelope glycoprotein gp41. The serum of some of the animals inoculated with v-env5 (Figure 10, lane a) also recognized gp50 and gp110, which indicates the ability of this recombinant virus to trigger an immune response to all LAV / HTLV III major glycoproteins.


 IMMUNOGENICITY OF V-ENV5 RECOMBINANT OF VACCINE-LAV / HTLV III VIRUS VIRUSES IN SUB-HUMAN PRIMATES
 



  The recombinant vaccinia-V-env5 immunogenicity of LAV / HTLV III was studied in two species of subhuman primates: Macaca Fasciculariès and chimpanzees. The results described below show that v-env5 is capable of eliciting humoral and cell-mediated immunity in both species.


 Humoral response in macaque monkeys immunized with v-env5
 



  Nine long-tailed macaques (Macaca Fascicularis), ranging from youth to adulthood, were used to study the immunogenicity of recombinant vaccinia virus v-env5. All animals were screened for the absence of vaccinia and LAV / HTLV III antibodies and the absence of monkey T-lymphotropic virus or monkey AIDS virus or antibodies against these viruses. Four of the monkeys were inoculated with 2 x 10 <8> v-env5 and four monkey plate forming units inoculated 2 x 10 <7> plaque forming units of the same virus. 2 x 10 were inoculated on an animal <7> plaque-forming units of a control recombinant vaccinia virus (recombinant vaccinia herpes simplex gD1 v-HSgD1). All animals were inoculated by scarifying the skin.

  Serum and heparinized blood samples were collected before inoculation, and after 4, 6 and 8 weeks after inoculation. Ten weeks after the first inoculation, all animals were given a second 2x10 inoculation <8> plaque forming units of the same virus. All animals showed self-healing of the skin lesions, typical of vaccinia virus infections, and normal physiological indications throughout the course of the experiment. The humoral response was analyzed by ELISA and by Western-type coagulation assay tests.



  For an "ELISA" analysis, the serum samples were diluted 50-fold in PBS containing 3% of non-fat milk powder and 0.2% of NP-40 (polyoxyethylene (9) p-tert-octylphenol), and they were reacted with LAV / HTLV III virion proteins which had been immobilized on plate troughs for microtiter. The procedure for ELISA was identical to that described above, except that the second antibody used was anti-human goat antibody conjugated to horseradish peroxidase.



  The results obtained, presented in FIG. 11, show that, if only two animals presented seroconversion after primary inoculation, all the animals presented seroconversion after the second immunization.



  To determine which antibodies were produced in the immunized animals, serum samples collected after 4 weeks after the second immunization were analyzed by Western-type coagulation. Two protocols were used for the optimal detection of the two envelope glycoproteins, gp41 and gp110. For optimal detection of gp41, the serum was diluted 50 times in PPS + 3% unpasteurized milk and 0.2% NP-40, and it was reacted for 1 hour at room temperature with LAV / HTLV III virion protein purified and immobilized on a nitrocellulose filter.

  Filters were washed with PBS (physiological saline supplemented with phosphate buffer) + 0.2% NP-40, and reacted with conjugated goat anti-human immunoglobulin antibody with alkaline phosphatase (Zymed, CA) at a dilution of 1: 2000 in PBS buffer + 0.2% NP-40. For the detection of the gp110 glycoprotein, the serum was diluted 50 times in PBS + 3% unpasteurized milk and reacted for 1 hour at room temperature with purified LV / HTVL III virion protein. and immobilized on a nitrocellulose filter. Filters were washed in PBS + 0.05% "Tween-20" and then reacted with goat anti-human immunoglobulin antibody conjugated with alkaline phosphatase (Zymed, CA) at dilution from 1 to 2000 in PBS buffer.

   In each procedure, the final washing after the second reaction with the antibody was carried out in 0.1 M TRIS (pH 9.5) with 0.1 M NaCl and 5 mM MgCl2. Antibody bound to antigens was detected on the filter by reacting the filter with a solution containing 0.1M Tris (pH 9.5), 0.1M NaCl, 5mM MgCl2, 0.33 g / ml bromo-chloro-indolyl phosphate and 0.17 mg / ml nitrotetrazolium blue. After reacting the filters with the chromogens, the filters were rinsed in water, air dried and photographed.



  As shown in Figure 12B, all animals that received two inoculations showed a strong antibody response to gp41. In addition, four of these animals also produced antibodies which reacted strongly with gp110. Taken as a whole, the results represented in FIG. 12A show that recombinant v-env5 is capable of eliciting, in macaque monkeys, the production of antibodies specific for the envelope of LAV / HTLV III.


 CELL-MEDIATED IMMUNOLOGICAL RESPONSE IN MACAQUES IMMUNIZED BY V-ENV5
 



  Peripheral blood lymphocytes were isolated from heparinized blood of macaques, obtained four weeks after a second intradermal immunization with v-env5, or a recombinant control vaccinia virus expressing herpes simplex virus glycoprotein D (v-HSVgD1) , by centrifugation on a "Ficoll Hypaque" device. Peripheral blood lymphocytes from macaque 81, which was vaccinated only once with v-env5, and also from non-immunized macaques were also isolated. The peripheral blood lymphocytes were suspended in "RMPI 1640" medium (Gibco, Grand Island, New York, United States of America) supplemented with 10% of normal human serum inactivated by heat and placed 1 x 10 <5> peripheral blood lymphocytes, in a final volume of 0.1 ml of the medium, in plate recesses comprising 96 round bottom recesses.

  0.1 ml of medium containing v-env5 inactivated by light (ultraviolet) (1 × 10) was introduced into each hollow <6> plaque forming units / ml before inactivation by ultraviolet) or unbroken LAV / HTLV III (1 mg / ml, approximately 1 x 10 <5> average infectious doses obtained by culture on tissue), which were purified by two cycles of centrifugation with a sucrose gradient to separate the supernatants from CEM cells infected with LAV / HTLV III, which is a cell line of HLADR-type T negative leukemia.

  Six days after stimulation, each hollow of peripheral blood lymphocytes was labeled with 1 mu Ci of 3H-TDR (3H-thydimine, New England Nuclear, Boston, MA, United States of America) for 6 hours, harvested cells, and counts per minute (cpm) corresponding to 3H-TDR incorporated were determined to be the average value obtained for four pits.

  The stimulation index was calculated by dividing the number of counts per minute corresponding to 3H-TDR incorporated into stimulated cells, by the number of counts per minute corresponding to the incorporation into unstimulated cells.
 <tb> <TABLE> Columns = 7
 <tb> Title: Table II
 <tb> Head Col 01 to 07 AL = L: Response, by LAV / HTLV III-induced proliferation, of peripheral blood lymphocytes taken from macaques, after immunization with a recombinant vaccinia virus expressing the envelope glycoproteins of LAV / HTLV III
 <tb> SubHead Col 01 AL = L> Macaque
not <o>:
 <tb> SubHead Col 02 AL = L> Immunization:
 <tb> SubHead Col 03 AL = L> None,
cpm *:
 <tb> SubHead Col 04 to 07 AL = L: Virus used to stimulate lymphocytes
 <tb> SubHead Col 04 to 05 AL = L:

  LAV / HTLV III
 <tb> SubHead Col 06 to 07 AL = L: v-env5
 <tb> SubHead Col 04 AL = L> cpm *:
 <tb> SubHead Col 05 AL = L> IS **:
 <tb> SubHead Col 06 AL = L> cpm *:
 <tb> SubHead Col 07 AL = L> IS **:
 <tb> <SEP> 67 <SEP> v-env5 <SEP> 1586 <SEP> 7465 <SEP> 4.7 <SEP> 60,415 <SEP> 38.1
 <tb> <SEP> 68 <SEP> v-env5 <SEP> 2245 <SEP> 9075 <SEP> 4.0 <SEP> 28,638 <SEP> 12.8
 <tb> <SEP> 74 <SEP> v-env5 <SEP> 1585 <SEP> 4732 <SEP> 3.0 <SEP> 21,487 <SEP> 13.6
 <tb> <SEP> 75 <SEP> v-env5 <SEP> 581 <SEP> 8645 <SEP> 14.9 <SEP> 37,847 <SEP> 14.1
 <tb> <SEP> 76 <SEP> v-env5 <SEP> 2479 <SEP> 5987 <SEP> 2.5 <SEP> 36,657 <SEP> 14.8
 <tb> <SEP> 80 <SEP> v-env5 <SEP> 1077 <SEP> 13,922 <SEP> 12.9 <SEP> 24,752 <SEP> 23.0
 <tb> <SEP> 81 <SEP> v-env5 <SEP> 965 <SEP> 3985 <SEP> 4.1 <SEP> 40,572 <SEP> 42.0
 <tb> <SEP> 82 <SEP> v-env5 <SEP> 2581 <SEP> 8580 <SEP> 3.3 <SEP> 46,847 <SEP> 18.1
 <tb> <SEP> 73 <SEP> V-HSVgD1 <SEP> 612 <SEP> 553 <SEP> 1.0- <SEP> 56,217 <SEP> 91.9
 <tb> <SEP> 26 <SEP> none

    <SEP> 2911 <SEP> 1822 <SEP> 1.0- <SEP> 2240 <SEP> 1.0-
 <tb> <SEP> 27 <SEP> none <SEP> 1228 <SEP> 1072 <SEP> 1.0 <SEP> 2532 <SEP> 2.0
  * cpm: strokes per minute corresponding to <3> H-TdR Incorporated
** IS: stimulation index
  
 <tb> </TABLE>



  As seen in Table II, all immune macaques, including number 81, which received a single inoculation of v-env5, produced lymphocytes which proliferated in specific response to LAV / HTLV III stimulation in vitro. In contrast, lymphocytes from animal 73, which received vaccinia-v-HSVgD1 recombinant herpes simplex, responded only to stimulation by vaccinia virus, but not to that by LAV / HTLV III. Unimmunized control animals did not produce lymphocytes responding to either antigen. Furthermore, the response for lymphocytes from macaques immunized with v-env-5 was similar in amplitude to that obtained for macaque 73, which was vaccinated with v-HSVgD1.

  These results indicate that expression of the LAV / HTLV III env gene by the recombinant virus did not suppress T cell-mediated immune responses in macaques in vitro or in vivo.



  To determine the subgroup of lymphocytes from immunized macaques that was stimulated by LAV / HTLV III, the stimulated peripheral blood lymphocytes were subjected to a two-color immunofluorescence examination for expression of the IL-2 receptors which are present on activated T cells and on B cells. The CD4 and CD8 antigens are present on the T cells and the CD20 antigen (Bp35) is present on the B cells. The results presented in Table III show that almost all of the blood lymphocytes peripherals stimulated by a virus, which express IL-2 receptors, also express CD4 or CD8 antigens, while only 1.5 to 2% coexpress the CD20 antigen (Bp35).

   This result indicates that the stimulated lymphocytes are T cells.
 <tb> <TABLE> Columns = 6
 <tb> Title: Table III
 <tb> Head Col 01 to 06 AL = L: Expression of IL-2 receptors and CD4, CD8 or CD20 (Bp35) antigens on peripheral blood lymphocytes obtained from vaccinated macaques, after stimulation with LAV / HTLV III or by a recombinant vaccinia virus expressing the envelope glycoproteins of LAV / HTLV III
 <tb> SubHead Col 01 AL = L> Macaque
not <o>:
 <tb> SubHead Col 02 AL = L> Lymphocytes * stimulated by:
 <tb> SubHead Col 03 AL = L>% total IL-2R + cells:
 <tb> SubHead Col 04 to 06 AL = L:% of IL-2R cells + coexpressing
 <tb> SubHead Col 04 AL = L> CD4:
 <tb> SubHead Col 05 AL = L> CD9:
 <tb> SubHead Col 06 AL = L> CD20 (Bp35):

   
 <tb> <SEP> 74 <SEP> v-env5 <SEP> 27.7 <SEP> 48.6 <SEP> 52.2 <SEP> 2.0
 <tb> <SEP> 80 <SEP> v-env5 <SEP> 25.6 <SEP> 62.4 <SEP> 40.0 <SEP> NT
 <tb> <SEP> 74 <SEP> LAV / HTLV III <SEP> 14.7 <SEP> 50.0 <SEP> 59.0 <SEP> 1.9
 <tb> <SEP> 80 <SEP> LAV / HTLV III <SEP> 12.0 <SEP> 58.0 <SEP> 38.0 <SEP> 1.5
 <tb> <SEP> 80 <SEP> no virus <SEP> 0.3 <SEP> 0.3- <SEP> 0.3- <SEP> 0.3-
 * lymphocytes: peripheral blood lymphocytes
** NT: no test
  
 <tb> </TABLE>



  The results of Table III were obtained as follows. Peripheral blood lymphocytes were stained simultaneously with 2A3 monoclonal antibody conjugated to phycoerythrin (Becton-Dickinson, Inc., Mountain View, CA, USA), antibody to the interleucine receptor -2 (IL-2R), and with fluorescein-conjugated IF5 monoclonal antibodies (Genetic Systems Corp., Seattle, WA, United States of America) for CD20, G10-1 (Genetic Systems Corp., Seattle, WA) for CD8 or T4a (Ortho Diagnostics, Raritan, NJ) for CD4. Antibodies were used at saturation concentrations, determined beforehand by titrations on lymphocytes analyzed by flow microfluorometry with a modified FACS IV classifier (Becton-Dickinson, Mountain View, CA).

  Two-color quantitative analysis was performed as previously indicated (Ledbetter, J.A. et al., 1984, Perspectives in Immunogenetics and Histocompatibility, 6, 119-129). Dispersion gates were set forward and at right angles to include lymphoblasts and a large number of small lymphocytes. The values indicated are those relating to the total percentage of IL-2R + cells and for the percentage of IL-2R + cells coexpressing CD4, CD8, or CD20 (Bp35).



  It is well established that T cells, after antigen simulation, produce lymphokines comprising IL-2, which can promote differentiation and / or proliferation of T and B cells and which can activate natural lytic cells or globules capable of lysing cells infected with various viruses, including the AIDS virus. We therefore wondered whether T cells or cells from vaccine macaques produce IL-2 after stimulation with LAV / HTLV III.



  To answer this question, we conducted two experiments. For Experiment 1, peripheral blood lymphocytes were isolated from heparinized blood of macaques four weeks after a second intradermal immunization injection with v-env5 or v-HSVgD1 constructed with the WR strain of the vaccinia virus. For experiment 2, peripheral blood lymphocytes were isolated from macaques 4 weeks after a first intradermal immunization injection by 2 × 10 <5> plaque-forming units of v-env5, v-HSVgD1 or a recombinant v-env5 virus constructed with the vaccine virus strain of the "New York City Board of Health" New York) (v-env5NY).

  Peripheral blood lymphocytes were suspended in RPMI 1640 medium supplemented with 10% normal heat-inactivated human serum and 2 x 10 were placed <5> peripheral blood lymphocytes in plaque pits with 96 round bottom pits. Was then added in the hollow v-env5 inactivated by ultraviolet light (1 x 10 <6> plaque forming units / ml before inactivation by ultraviolet light) or purified LAV / HTLV III (1 mu g / ml). Two days after stimulation, the supernatants were harvested from pits used in pairs and tested for the ability to maintain proliferation of IL-2 dependent CTLL-2 cells (cells supplied by Dr. S. Gillis, Immunex Corp. Seattle, WA, United States of America), which were washed for 24 hours before testing to rid them of IL-2.

  During the last six hours of incubation with the supernatant, the cells were labeled with <3> H-TdR, and the amount of <3> H-TdR incorporated into cells. The activity units of IL-2 present in the supernatant liquid were calculated, as described, from reference curves obtained by tests of the effect of recombinant human IL-2 (supplied by Dr Gillis) on the proliferation of CTLL-2 cells.

  The results obtained are presented in Table IV.
 <tb> <TABLE> Columns = 5
 <tb> Title: Table IV
 <tb> Head Col 01 to 05 AL = L: Production of IL-2 by peripheral blood lymphocytes from vaccinated macaques, after stimulation by LAV / HTLV III or by recombinant vaccinia viruses expressing envelope glycoprotein from AIDS virus
 <tb> Head Col 01 to 02 AL = L: Experiment 1:
 <tb> Head Col 03 to 05 AL = L: IL-2 (units / ml)
 <tb> SubHead Col 01 AL = L> Macaque
not <o>:
 <tb> SubHead Col 02 AL = L> Immunization:
 <tb> SubHead Col 03 to 05 AL = L:

  Supernatants of lymphocytes * stimulated by
 <tb> SubHead Col 03 AL = L> no virus:
 <tb> SubHead Col 04 AL = L> LAV / HTLV III:
 <tb> SubHead Col 05 AL = L> v-env5:
 <tb> <SEP> 67 <SEP> v-env5 <SEP> 0 <SEP> 28.0 <SEP> 96.0
 <tb> <SEP> 68 <SEP> v-env5 <SEP> 0 <SEP> 16.0 <SEP> 124.0
 <tb> <SEP> 74 <SEP> v-env5 <SEP> 0 <SEP> 9.0 <SEP> 52.0
 <tb> <SEP> 73 <SEP> v-HSVgD1 <SEP> 0 <SEP> 0 <SEP> 108.0
 <tb> <SEP> 26 <SEP> none <SEP> 0 <SEP> 0 <SEP> 0
 <tb> Head Col 01 to 05 AL = L: Experiment 2:
 <tb> <SEP> 03 <SEP> v-env5 <SEP> 0 <SEP> 14.4 <SEP> 55.8
 <tb> <SEP> 05 <SEP> v-env5NY <SEP> 0 <SEP> 16.8 <SEP> 33.3
 <tb> <SEP> 49 <SEP> v-env5NY <SEP> 0 <SEP> 7.1 <SEP> 26.7
 <tb> <SEP> 52 <SEP> v-env5NY <SEP> 0 <SEP> 2.4 <SEP> 27.6
 <tb> <SEP> 59 <SEP> v-HSVgD1 <SEP> 0 <SEP> 0 <SEP> 27.6
 <tb> <SEP> 26 <SEP> none <SEP> 0 <SEP> 0 <SEP> 0
 <tb> <SEP> 27 <SEP> none <SEP> 0 <SEP> 0 <SEP> 0
 <tb> </TABLE>



   The results of experiment 1 of table IV show that the supernatant cells originating from peripheral blood lymphocytes stimulated by v-env5 or by LAV / HTLV III obtained on macaques immunized twice with v-env5, contain IL-2, as shows their ability to induce proliferation of CTLL-2 cells, which is an IL-2 dependent cell line.

  Similarly, as shown in Experiment 2 of Table IV, IL-2 was detected in what supernatant peripheral blood lymphocytes stimulated by LAV / HTLV III or by v-env5 originating from macaque 03, which was immunized a times with v-env5, and coming from all three macaques which have been immunized once by the strain of "vaccines" of the same recombinant (v-env5NY, see above the presentation on the construction and characterization of the Recombinant vaccinia virus containing the chimeric envelope gene of LAV / HTLV III). In contrast, peripheral blood lymphocytes from macaques 73 and 59, which have undergone immunization inoculations with recombinant vaccinia-HSV gD-1 viruses, produced IL-2 after stimulation with v-env5 only, and not not after stimulation with LAV / HTLV III.

  The non-immunized macaques 26 and 27 did not produce detectable IL-2 after stimulation with LAV / HTLV III or with the recombinant vaccinia virus. Since IL-2 cells or T cells with auxiliary / inducing activity produce IL-2 after antigenic stimulation, these results show the presence of T helper cells or globules, which recognize LAV / HTLV III, in macaques immunized by recombinant viruses.

  In addition to their likely role in differentiating B cells or lymphocytes to produce antibodies against LAV / HTLV III envelope antigens, IL-2 producing cells or T lymphocytes may be involved in differentiation and / or expansion of effector cells, such as cytotoxic T lymphocytes or natural lytic cells which can kill virus infected cells.


 HUMOR RESPONSES IN CHIMPANZES IMMUNIZED BY V-ENV5NY
 



  5 x 10 were injected intradermally into two juvenile chimpanzees <8> v-env5NY plaque forming units, the v-env5 "vaccine" strain. An animal was intradermally inoculated with the same titer of a vaccinia-herpes simplex gD recombinant, v-HSVgD1NY constructed from the same parental vaccine strain (v-NY) as v-env5NY. A second inoculation of the same dose was administered to all animals 8 weeks after the first immunization. The serum samples were collected twice a week after immunization and subjected to assays, by ELISA and Western-type coagulation, to look for antibodies specific for LAV / HTLV III.

  All animals exhibited self-healing of the skin lesions, typical of vaccinia virus infection, and had normal physiological results throughout the course of the experiment.



  The methods for an ELISA type examination on chimpanzee sera were identical to that used for macaque sera. The results presented in Table V indicate that the two experimental animals (124 and 149) underwent sero-conversion after 8 weeks after the first immunization and that the levels of antibodies continued to increase after the second injection of immunization.

  The control animal (134) remained seronegative.
 <tb> <TABLE> Columns = 4
 <tb> Title: Table V
 <tb> Head Col 01 to 04 AL = L: ELISA assay of serum samples from chimpanzees immunized with recombinant vaccinia viruses
 <tb> SubHead Col 01 AL = L> Sampling time:
 <tb> SubHead Col 02 to 04 AL = L:

  ELISA reading for LAV / HTLV III specific serum antibodies
 <tb> SubHead Col 02 AL = L> Chimpanzee n <o> 134 v-HSVgDINY:
 <tb> SubHead Col 03 AL = L> Chimpanzee n <o> 124 v-env5NY:
 <tb> SubHead Col 04 AL = L> Chimpanzee n <o> 149 v-env5NY:
 <tb> <SEP> Before bleeding <SEP> 0.084 +/- 0.015 <SEP> 0.100 +/- 0.005 <SEP> 0.123 +/- 0.025
 <tb> <SEP> 8 weeks after the first immunization injection <SEP> 0.085 +/- 0.010 <SEP> 0.185 +/- 0.020 <SEP> 0.403 +/- 0.027
 <tb> <SEP> 2 weeks after the second immunization injection <SEP> 0.075 +/- 0.012 <SEP> 0.237 +/- 0.017 <SEP> 0.523 +/- 0.073
 <tb> </TABLE>



  To show that the specific LAV / HTLV III antibodies detected in the immunized animals were directed against the envelope glycoproteins, the same sera were analyzed by Western-type coagulation. The procedure used has been optimized for the detection of the gp41 antibody, and it has been identical to that used for the analysis of macaque sera. The results presented in FIG. 13 show that the antibodies specific for LAV / HTLV III, produced in the two vaccinated animals, of course act against the envelope glycoproteins, and that the level of these antibodies increased after the second immunization injection.


 CELL-MEDIATED IMMUNOLOGICAL RESPONSES IN CHIMPANZES IMMUNIZED BY V-ENV5NY
 



  Peripheral blood lymphocytes, isolated from the same animals as described above, were used to show the cell-mediated immunological responses in these animals. Peripheral blood lymphocytes were isolated from heparinized blood by Ficoll-hypaque type centrifugation 4 weeks after the initial intradermal inoculation of the recombinant vaccinia viruses. These lymphocytes were seeded at a rate of 1 x 10 <5> cells by trough in microtiter plates comprising 96 troughs, in RPMI 1640 medium supplemented with 10% of normal human serum heat inactivated and penicillin / streptomycin. The LAV / HTLV III glycoproteins (5 mu g / ml) or LAV / HTLV III envelope glycoproteins (1 mu g / ml), isolated by lectin chromatography on LAV / HTLV III, were then added to the depressions. purified.

  Six days later, it was determined by scintillation counting in a liquid medium, H <3> -thymidine incorporated into cells.



   As shown in Table VI, lymphocytes from the two animals (no <S> 124 and 149) immunized with v-env5NY, proliferated in response to stimulation by the LAV / HTLV III virion or by purified envelope proteins (env). On the contrary, animal 134, which received vaccinia, herpes simplex recombinant v-HSgD1NY, did not produce lymphocytes recognizing LAV / HTLV III.
 <tb> <TABLE> Columns = 5
 <tb> Title: Table VI
 <tb> Head Col 01 to 05 AL = L: Cell-mediated immunological response in chimpanzees vaccinated with vaccinia recombinants
 <tb> SubHead Col 01 AL = L> Chimpanzee
not <o>:
 <tb> SubHead Col 02 AL = L> Immunization:
 <tb> SubHead Col 03 to 05 AL = L:

   <3> H-thymidine incorporated into lymphocytes * stimulated by
 <tb> SubHead Col 03 AL = L> no antigen:
 <tb> SubHead Col 04 AL = L> LAV / HTLV III:
 <tb> SubHead Col 05 AL = L> approx:
 <tb> <SEP> 124 <SEP> v-env5NY <SEP> 2842 <SEP> 37,132 <SEP> 26,370
 <tb> <SEP> 149 <SEP> v-env5NY <SEP> 375 <SEP> 20,292 <SEP> 27,115
 <tb> <SEP> 134 <SEP> v-HSVgD1NY <SEP> 367 <SEP> 1987 <SEP> 367
 <tb> <SEP> 64 <SEP> none <SEP> 452 <SEP> 567 <SEP> 222
 <tb> <SEP> 72 <SEP> none <SEP> 737 <SEP> 2417 <SEP> 905
 * peripheral blood lymphocytes
  
 <tb> </TABLE>



  Peripheral blood lymphocytes from all chimpanzees exhibited high titers of <3> H-thymidine (38,870 to 109,790 cpm) after stimulation with phytohemagglutinin (PHA, 2 mu g / ml) and high levels of incorporation of <3> H-thymidine (42,172 at 12,067 cpm) after stimulation with xenogenous human peripheral blood lymphocytes, grouped, having undergone X-ray irradiation. Peripheral blood lymphocytes from chimpanzees immunized with v-env5 and also the chimpanzee immunized with v-HSVgD1NY, showed strong proliferation responses to vaccinia viruses, while peripheral blood lymphocytes from non-immunized chimpanzees did not proliferate in response to stimulation by the virus vaccinated.



  Overall, the results presented in Tables I to VI and in Figures 11 to 13 indicate that (a) the recombinant vaccinia virus v-env5 and its counterpart in the vaccine strain, v-env5NY, were able to elicit humoral and cell-mediated immunological responses specific for LAV / HTLV III in two species of subhuman primates, macaques and chimpanzees, and (b) that the recombinant viruses have not suppressed the immunological responses mediated by T cells or lymphocytes, in vitro or in vivo.


 DECREASE IN NEUROTOXICITY OF RECOMBINANTS CONSTRUCTED WITH THE V-NY STRAIN OF THE VACCINE VIRUS
 



  Groups of five mice (ICR strain) were injected into the brain with aliquots of virus, containing 5 x 10 <7> plaque forming units in 25 to 50 mu l. We rated the reported 10-day mortality report after inoculation.
 <tb> <TABLE> Columns = 2
 <tb> Title: Table VII
 <tb> Head Col 01 to 02 AL = L: Mortality of mice having undergone intracerebral inoculation with vaccinia recombinants
 <tb> SubHead Col 01 AL = L> Inoculation:
 <tb> SubHead Col 02 AL = L> Dead animals /
animals treated by injection:
 <tb> <SEP> v-NY <SEP> 0/5
 <tb> <SEP> v-env5NY <SEP> 0/5
 <tb> <SEP> n-env2NY <SEP> 0/5
 <tb> <SEP> v-env7NY <SEP> 0/5
 <tb> <SEP> smallpox vaccine (Wyeth) <SEP> 3/5
 <tb> <SEP> v-env5 <SEP> 5/5
 <tb> <SEP> v-env2 <SEP> 5/5
 <tb> <SEP> v-env7 <SEP> 5/5
 <tb> <SEP> saline solution <SEP> 0/5
 <tb> </TABLE>



  The results presented in Table VII indicate that the vaccinia virus originating from a tissue culture, purified on a plate (strain v-NY) and its derivatives v-env5NY, v-env2NY and v-env7NY, have reduced neurotoxicity in comparison of the WR strain and its derivatives. Since the New York City Board of Health strain was used as a smallpox vaccine, recombinants based on this strain should be better suited for the development of a vaccine for use. human.


 EXAMPLE 2 GAG RECOMBINANTS OF BACULOVIRUS
 



  In the following example, various plasmid vectors were constructed containing chimeric genes comprising LAV / HTLV III gag coding sequences located downstream from the AcNPV transcription control sequences.



  These chimeric genes, containing the polyhedrin promoter AcNPV and the coding sequence gag of LAV / HTLV III, were inserted into the genome of AcNPV by recombination in vivo. Such recombinant viruses have been identified and purified, and virus stocks have been prepared from infected tissue culture cells. Immunoreactive proteins, related to LAV / HTLV III gag, have been shown to be produced in vitro by the recombinant AcNPV. Cells infected with recombinant viruses have been shown to exhibit positive immunofluorescence. Finally, lysates of cells infected with recombinant AcNPV were positive in the ELISA assay when tests were carried out with serum obtained from patients suffering from AIDS.

  A detailed description of each step of this embodiment of the invention is presented below.


 GENERAL OPERATING MODES
 


 Cells and viruses
 



  Cells of Spodoptera frugiperda, clone Sf9, were obtained from the American Type Culture Collection (ATCC n <o> CRL 1711) and its propagation was carried out in Antheraea de Grace medium (Gibco, KC Biologicals) containing 3.3 g / l of a yeast hydrolyzate (from Difco) and 3.3 g / l of a lactalbumin hydrolyzate (Difco), 10% of fetal bovine serum and 0.06 g / l of penicillin as well as 0.1 g / l of streptomycin (TNM-FH medium). The cells were grown at 28 DEG C. The Autographa californica nuclear polyhedrosis virus was obtained from Dr Lois Miller (Department of Genetics and Entomology, University of Georgia, Athens, GA 30602, United States of America) and this virus was purified by plate on Sf9 cells containing 1% agar and 0.8 × TNM-FH. Virus stock dilutions were made in TNM-FH.


 DNA PREPARATION, RESTRICTION AND MODIFICATIONS
 



  Restriction enzymes were purchased from Bethesda Research Laboratories Inc., and the conditions for restriction digests were those suggested by the supplier. The Kenow fragment of DNA polymerization of E. coli was used at 200 units / ml in 50 mM Tris-HCl (pH 7.4), 6 mM MgCl2, 10 mM 2-mercaptoethanol at 37 DEG C for 30 minutes.



   Viral DNA was prepared for transfection from wild type virus non-occluded virus (WNV) stocks, using the following procedure of Miller, L.K; Miller DW and Safer, P .: a pellet of WNV particles was obtained to separate them from the supernatant liquid through a cushion of 25% sucrose, 5 mM NaCl and 10 mM EDTA, by centrifugation at 90,000 g for 1 hour at 5 DEG C. The supernatant was removed and the virus pellet was resuspended in 0.5 ml of lysis buffer (10 mM Tris, pH 7.6, 10 mM of EDTA, 0.25% SDS). After resuspending the virus pellet, proteinase K was added to a final concentration of 500 µg / ml and incubated overnight (about 16 hours) at 37 DEG C with occasional shaking.

  The DNA was extracted with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1). The DNA was then precipitated at -20 DEG C by the addition of 1/10 volume of 3M sodium acetate at pH 5 and 2 volumes of cold ethanol. After obtaining a centrifugation pellet, the DNA was washed with 70% ethanol, dried and resuspended in TE (10 mM Tris, 1 mM EDTA, pH 7.5 ) before using it for transfection or restriction enzyme analysis.



  Plasmid DNA was prepared using the procedure of Summers, MD and Smith, GE as follows: A single colony of bacterial cells, from a freshly prepared plate, was inoculated with 1 ml of Luria broth ( BL) supplemented with an appropriate antibiotic. After incubation at 37 DEG C for 12 to 18 hours, the ml of culture was transferred into 200 ml of Luria broth plus antibiotics in a 500 to 1000 ml flask, and incubated in a shaking incubator ( 37 DEG C) overnight (12 to 6 p.m.). Cultures, 200 ml each, were transferred to centrifuge bottles, and centrifuged at 2,500 rpm for 10 minutes.

  After removal of the supernatant, each cell pellet was resuspended in 30 ml of STET buffer (8% sucrose, 0.5% Triton X-100, 50 mM EDTA (pH 8.0), 10 mM of Tris-Cl, pH 8.0) at room temperature and transferred to a 125 ml flask. Then 3 ml of 10 mg / ml of lysozyme (prepared fresh in STET buffer) were added to each flask, and each flask was swirled to mix, then incubated for about 5 minutes at room temperature. Each balloon was then gently swirled (about once every 5 seconds) directly over a flame until the cells began to clot and turn white.

  When bubbles formed, the flasks were transferred to a boiling water bath for 45 seconds, after which the flasks were cooled in an ice water bath for 2 minutes or more. Viscous mixtures were slowly poured into 50 ml round bottom centrifuge tubes and centrifuged for 15 minutes at 16,000 rpm using a rotor SW 28 (Beckman, CA) in a preparation centrifuge . The supernatant from each tube was carefully poured into a polypropylene centrifuge tube, discarded after use, to which was added one volume of isopropanol or two volumes of ethanol, and the DNA was precipitated to -80 DEG C for 20 minutes (or -20 DEG C for 2 hours or more), and centrifuged at 2500 g for 15 minutes or more.

  After removing the alcohol, 2.5 ml of extraction buffer were added (per liter: 12.1 g of Tris, 33.6 g of Na2EDTA, 2H2O, 14.9 g of KCl, pH 7.5) at the pellet, which was then swirled to detach the lysate from the bottom of the tube. 100 µl of 10 mg / ml RNase A (dissolved in 0.1X TE and pretreated with 10 minutes of boiling) was added and incubated for 30 minutes at 37 DEG C. It was added to each tube approximately 200 μg of proteinase K and then incubated at 40-50 DEG C for approximately 30 minutes, at the end of which 250 μl of 10% "Sarkosyl" was added and the reaction was continued. incubation for at least 3 hours. In order to prepare CsCl gradients, 80 μl of 10 mg / ml ethidium bromide per ml of DNA solution were added to each tube.

  Then 1.04 g of CsCl per ml of DNA solution was added to each tube and gently vortexed to dissolve. After transfer to "Quick Seal" tubes <(R)>, the gradients were centrifuged until equilibrium at 55,000 rpm for 16 hours in a 70.1 Ti fixed angle rotor, which gave two visibly well separated bands of DNA in which the upper band comprises the linear and notched DNA while the lower band comprises circular DNA covalently closed. The lower band (circular DNA covalently closed) was collected and poured into a 15 ml centrifuge tube, made of polypropylene, in which water was added to bring the volume to about 6 ml in each tube. Ethidium bromide was extracted from DNA, using an equal volume of isoamyl alcohol, and the pink phase (upper) was removed and discarded.

  The extraction was repeated until the upper phase was colorless, and the lower phase (DNA) was clear and colorless. There must remain in each tube approximately 5 ml of DNA solution (otherwise, water is added to a final volume of 5 ml) to which 2 volumes of absolute ethanol have been added. The DNA was precipitated at -20 DEG C overnight or at -80 DEG C for 10 minutes, and a pellet was centrifuged at 2500 g for 20 minutes. After removing all of the alcohol, 500 μl of 0.1X TE was added to each pellet, and the pellet was resuspended at 65 DEG C for at least 10 minutes. DNA can be stored at 4 DEG C.



  Plasmid DNA was also prepared as described in Maniatis et al., 1982, Molecular Cloning, Cold Spring Harbor Laboratory (page 8696). The DNA ligase was purchased from New England Biolabs, and used at 10,000 units // HTLV III gag in the vaccinia virus genome, by in vivo recombination. Such recombinant viruses have been identified and purified, and virus stocks have been prepared from cells obtained by culture on infected tissue. Immunoreactive proteins related to LAV / HTLV III gag have been shown to be produced in vitro by recombinant vaccinia viruses. A detailed description of each step in this embodiment of the invention is presented below.

   The general procedures used for this embodiment have been described above with respect to vaccinia envelope recombinants.


 Construction of vector plasmids containing a vaccinia virus promoter, ligated to the coding sequences of the LAV / HTLV III gag gene
 



  Five vector plasmids were constructed which contained the 7.5K promoter of vaccinia virus ligated to various lengths of the LAV / HTLV III genome containing gag coding sequences. The source of the LAV / HTLV III gag coding sequences were two related lambda J19 subclones (Wain-Hobson et al, Cell 40: 1985), pKS- and pSS-5. Plasmid pKS-5 contains a fragment of 3148 base pairs ranging from SStI to KpnI (nucleotide n <o> 224 to n <o> 3372) of LAV / HTLV III DNA inserted at the corresponding sites of pUC18. Plasmid pSS-5 contains a fragment of 5107 base pairs from SstI to Sa1I (nucleotide no 224 to 5221) of LAV / HTLV III DNA inserted at the corresponding sites of pUC18. Various lengths of gag coding sequences from pKS-5 or pSS-5 were inserted into plasmid pGS62, which is identical to PGS20 (Mackett et al., 1984, J.

  Virol. 49: 857-864), except that it has a single EcoRI site located downstream from the single SmaI site (see Figure 20). The specific sequences of LAV / HTLV III, inserted into pGS62, all start with a ThaI site (FnuDII) located 76 base pairs upstream of the start codon of the gag gene. Plasmids pv-gag1, pv-gag2, pv-gag3, pv-gag4 and pv-gag5 contain specific sequences of LAV / HTLV III ranging from this ThaI site to EcoRI (at 4194), KpnI (at 3372) sites , EcoRv (at 2523), BclI (at 1975) or BglII (at 1642), respectively. The description of the construction of each plasmid, carried out below, is made easier when reference is made to FIG. 20.



  Construction of pv-gag1: 5 μg of the plasmid DNA pSS-5 were digested until completion with restriction enzymes ThaI EcoRI. The resulting fragments were resolved on a 1% LMP agar gel. A 3.9 kbp fragment containing the LAV / HTLV III gag coding sequences was isolated and ligated to pGS62 DNA previously digested with SmaI and EcoRI. The ligation mixture was used to transform E. coli HB101 and clones able to resist ampicillin were chosen. Plasmids from individual colonies were tested for the presence of the inserted 3.9 kilopair base (kbp) fragment, by restriction analysis and by regeneration of the EcoRI site at the ligation junction.

  The resulting construct contains the entire coding sequence of the gag and protease (prt) genes, and a major part of the pol open reading frame, up to the EcoRI site at nucleotide 4194.



  Construction of pv-gag2: 5 μg of the plasmid pKS-5 were digested, until completion, with restriction enzymes ThaI and SmaI. The SmaI site of pKS-5 is located near the KpnI site, which is the junction between the sequence inserted from LAV / HTLV III and the multiple cloning sites of the plasmid pUC18. The resulting fragments were resolved on a 1% LMP agar gel, and a 3.2 kbp fragment containing sequences corresponding to LAV / HTLV III gag was isolated, and ligation was carried out on pGS 62 DNA, previously subjected to digestion with SmaI and treated with CIAP. The ligation mixture was used to transform E. coli HB101 and clones able to resist ampicillin were chosen.

  Plasmids from individual colonies were tested for the presence of the 3.1 kilopair base fragment and the orientation of the insertion was determined by restriction analysis. The resulting construction pv-gag2, contains the entire gag gene, the prt gene and part of the open reading frame pol up to the KpnI site at nucleotide 3372.



  The construction of pv-gag3 was similar to that of pv-gag2, except that a 2.3 kbp fragment, generated by ThaI and EcoRV digests of pKS-5, was used for site insertion. SmaI from pGS62. The final construct, pv-gag3, contains all of the gag and prt genes and a small portion of pol fused to the 3 min portion of the vaccinia virus TK gene.



  Construction of pv-gag4: 5 μg of plasmid DNA pSS-5 were subjected to digestion, until completion, with SStI and BclI. The resulting fragment was purified, containing the sequence corresponding to LAV / HTLV III gag. This fragment was ligated to a vector plasmid pIC19R (Marsh et al., 1984, gene 32: 481-485) previously cut with SstI and BamHI to construct an intermediate plasmid. The insertion of the gag sequence at the BamHI site of pIC19R results in the juxtaposition of the BclI site in the LAV / HTLV III sequence on the EcoRI site in this cloning vector with multiple sites. The digestion of this intermediate construction by the restriction enzymes ThaI and ECoRI gave rise to a fragment of 1.72 kilopairs of bases (kbp) which could easily be inserted into the plasmid pGS62 at the SmaI and EcoRI sites.

  The final construct, pv-gag4, contains the entire LAV / HTLV III gag gene and part of the prt gene fused to the 3 min part of the vaccinia virus TK gene.



  The construction of the plasmid pv-gag5 used the same two-step strategy as for pv-gag4. 5 μg of pSS-5 were digested with restriction enzymes SstI and Bg1II. A 1.42 kbp fragment containing the LAV / HTLV III gag sequence was isolated, and this fragment was purified on a 1% LMP agar gel and inserted into plasmid pIC19R at SstI and BglII sites. Ligation of the gag sequence at the BglII site on its corresponding site in pIC19R allows (a) the formation of a stop codon in phase with the open gag reading frame, and (b) the juxtaposition of the Bg1II site on the site adjacent EcoRI on the plasmid with multiple cloning sites. This intermediate construction was then subjected to digestion with ThaI and EcoRI.

  A fragment, 1.39 kilopair of bases containing the sequences corresponding to LAV / HTLV III gag, was purified, and this fragment was ligated to the SmaI and EcoRI sites on the plasmid pGS62. The resulting plasmid, pv-gag5, contains most, but not all, of the gag coding sequence (up to the Bg1II site) ligated to a frame translation stop sequence.


 Construction of recombinant vaccinia viruses containing chimeric LAV / HTLV III gag genes
 



   The insertion of chimeric LAV / HTLV III gag genes originating from vector plasmids pv-gag1, pv-gag2, pv-gag3, pv-gag4 and pv-gag5, into the genome of the vaccinia virus, was carried out by the same protocol than that used for the construction of v-env5 and v-env2 (see above). In the examples which follow, all the recombinant viruses were constructed using the strain, purified by plaques, from the "New York City Board of Health" (v- NY, see above). We chose TK recombinants <-> and they were purified on a plate three times before carrying out the expansion into stocks of virus which were used for subsequent characterizations.

  The recombinant viruses from pv-gag1, pv-gag2, pv-gag3, pv-gag4 and pv-gag5 were called v-gag1NY, v-gag2NY, v-gag3NY, v-gag4NY and v-gag5NY, respectively.


 Expression of LAV / HTLV III GAG-related proteins in tissue culture cells infected with recombinant vaccinia viruses
 



  The recombinant vaccinia viruses carrying the chimeric LAV / HTLV III gag gene, described above, are capable of expressing proteins related to LAV / HTLV III gag, when they are introduced by infection into tissue culture cells. Some of these recombinants have been able to pass the protein corresponding to precursor gag, p55, to the state of the mature proteins p25 and p18. These proteins have been found to be immunoreactive with serum from AIDS patients and / or with monoclonal antibodies against gag p25 or p18 proteins.



  Infected, at an infection rate of 10, confluent BSC-40 cells, in a 60 mm dish, by parental type vaccinia virus (v-NY) or by its recombinant derivatives, v-gag1NY, v-gag2NY, v-gag3NY, v-gag4NY or v-gag5NY. The infection was allowed to continue for 12 hours, after which the cells were harvested, washed once with phosphate buffered saline and collected by centrifugation. The centrifuged pellets of infected cells were resuspended in 0.3 ml of Laemmli sample buffer and lysis was carried out by 4 minutes of boiling. The entire cellular protein was resolved in 20 µl of cell lysate by electrophoresis on a 15% polyacrylamide-SDS gel.

  A purified LAV / HTLV III virion sample and an aliquot of a lysate of cells infected with non-infectious elements ("mock") were included as controls.



  The contents of the gel were electrophoresed onto a sheet of a nitrocellulose filter. The filter was first reacted with serum from AIDS patients, or with a combination of monoclonal antibodies against the gag proteins p25 and p18. After extensive washing, the filter was then reacted with goat anti-human immunoglobulin antibodies conjugated on alkaline phosphatase (AP conjugate) in cases where patient serum was used, or with AP conjugated goat anti-mouse immunoglobulin antibodies, in cases where mouse monoclonal antibodies have been used. The conditions for the first and second reactions with the antibodies as well as for the washings were as described above with regard to the identification of the related proteins in the envelope of LAV / HTLV III.

  The final washing after the second antibody reaction was carried out in 0.1M Tris (pH 9.5) with 0.1 M NaCl and 5 mM MgCl2. Antibody bound to antigens was detected on the filter by reacting the filter with a solution containing 0.1 M Tris (pH 9.5), 0.1 M NaCl, 5 mM MgCl2, 0, 33 mg / ml of bromo-chloro-indolyl phosphate, and 0.17 mg / ml of nitrotetrazolium blue. After the filters reacted with the chromogens, the filters were washed with water, air dried and photographed.



  The results of this analysis are presented in FIG. 21. The recombinants v-gag1NY and v-gag2NY contain all of the gag and prt genes. These recombinants express a family of proteins related to LAV / HTLV III which have co-migrated with the major gag proteins of LAV / HTLV III, p55, p40, p25 and p18. These proteins have been immunoreactive both with serum from AIDS patients (FIG. 21B) and with mouse monoclonal antibodies against p25 and p18 (FIG. 21A). The recombinant v-gag3NY contains the gag and prt genes, but its open reading frame pol is ligated in phase with the carboxy terminal part of the vaccinia TK gene. V-gag3NY was also able to express the primary gag gene product, p55.

  However, treatment of p55 appears to have been less effective than in the case of v-gag1NY and v-gag2NY, since there was much less p25 and p18, compared to p55, in lysates of cells infected with v-gag3NY. The recombinant v-gag4NY contains the entire gag gene, but only part of the prt gene. He synthesized a protein related to LAV / HTLV III similar in dimensions to p55. The recombinant v-gag5NY contains only part of the gag gene and expressed a truncated protein of 43 kD (Figure 21C). Although v-gag4NY and v-gag5NY do not appear to have effectively treated their primary gag-like proteins, their products have been immunoreactive against monoclonal antibodies against p25 and p18.

  In sum, despite the differences in processing capacity, the five recombinants were all capable of expressing immunoreactive proteins which contained the major epitopes of the LAV / HTLV III core proteins.


 EXAMPLE: BACULOVIRUS ENVELOPE RECOMBINANTS
 



  In the following example, a vector plasmid was constructed containing a chimeric gene comprising the LAV / HTLV III envelope coding sequences housed downstream relative to the AcNPV transcription control sequences. The chimeric gene contained the polyhedrin promoter AcNPV and the coding sequence of LAV / HTLV III env was inserted, by recombination in vivo, into the genome of AcNPV. The recombinant virus was identified and purified, and virus stocks were prepared from infected supernatants. LAV / HTLV III env related immunoreactive protein has been shown to be produced in vitro by the recombinant baculovirus. A detailed description of each step of this embodiment of the invention is presented below.

   The general procedures used for this embodiment are as described above for baculovirus recombinants.


 Construction of vector plasmids containing the AcNPV promoter ligated to the coding sequences of the LAV / HTLV III env gene
 



  The coding (or coding) sequence for the LAV / HTLV III env gene was purified from pv-env5 (see above, in the section on the construction and characterization of the recombinant vaccinia virus containing the chimeric gene). envelope of LAV / HTLV III), which was used for the construction of the recombinant vaccinia virus v-env5, which has been shown to express proteins related to env. The env coding region in pv-env, as well as the untranslated sequences of 96 base pairs neighboring 5 min and 223 base pairs neighboring 3 min, were surrounded at both ends by BamHI sites. A partial digestion of this plasmid, which also contained an internal BamHI site) using the restriction enzyme BamHI generated a 2.9 kbp fragment containing only specific LAV / HTLV III sequences.

  This fragment was resolved (about 0.5 µg) and purified from a 1% LMP agar gel, and ligation was performed on 0.5 µg of the vector plasmid pAc610, which had previously been linearized using BamHI and processed by CIAP. The ligation mixture was used to transform the MC1000 strain of E coli and the colonies resistant to ampicillin were chosen. Plasmid DNA of individual transformants was subjected to assays for the presence of LAV / HTLV III env sequences (i.e., the formation, by BamHI digestion, of 2.3 fragments Kbp and 0.54 Kbp) and to determine the orientation of the inserted part. The plasmid comprising the sequence corresponding to LAV / HTLV III env, inserted in the correct orientation, was purified and called pAc-env5.

  Its structure is shown in Figure 22.


 Construction of the recombinant baculovirus containing the chimeric LAV / HTLV III env gene
 



  The LAV / HTLV III env chimeric gene is inserted into the AcNPV genome by in vivo recombination, as explained above (construction of the recombinant baculovirus containing the LAV / HTLV III gag chimeric gene). The introduction by transfection of 1 mg of AcNPV DNA, 5 mg of pAc-env5 DNA and 15 mg of calf thymus DNA into Sf9 cells was carried out as described in the same above-mentioned paragraphs. After 5 days of incubation at 28 DEG C with 4 ml of complete medium, the supernatants were collected and clarified by centrifugation at 1000 g for 10 minutes. The virus stock was titrated on Sf9 cells, and the recombinant virus was first identified, by the plaque hybridization assay of M.D. Summers and G.E. Smith, as follows:



  Sf9 cells were seeded on 60 x 15 mm culture plates at a density of 2.5 x 10 <6> cells per plate in TNM-FH medium without serum. After fixing the cells, the medium was removed and 1 ml of a diluted virus inoculum was added to each plate. After 1 hour of incubation at 27 DEG C, the entire inoculum was removed from each plate and 4 ml of an upper layer of LMP agar was slowly added to the edge of each plate. Once the upper layer had solidified, the plates were incubated in a humid environment for 4 to 6 days, or until the colonies or plates of cells were well formed. The agar plates were then allowed to dry overnight or longer, leaving them in an unhumidified environment.

  When they were sufficiently dry, the upper agar layers were removed and a dry nitrocellulose filter was placed over the cells remaining in the culture plate. A 47 mm circle of 3M MM Whatman paper was saturated with solution A (0.05 M Tris-HCl, pH 7.4 and 0.15 M NaCl) and placed on top -the nitrocellulose filter in the culture plate. After absorption, the nitrocellulose filter was carefully removed and placed (cell side over) on Whatman filter paper saturated with solution B (0.5N NaOH, 1.5 M NaCl ). After 2-3 minutes, the nitro-cellulose filter was air dried on paper towels and transferred to Whatman filter paper saturated with solution C (1.0 M Tris-HCl , pH 7.4; 1.5 M NaCl).

  After 2-3 minutes, the nitrocellulose filter was air dried on paper towels, and washed by gentle immersion in a petri dish filled with solution D (0.3 M in NaCl, 0.03 M sodium citrate). The filter was then removed and dried on paper towels, and heated to 80 DEG C for 2 hours under vacuum. The filter was fixed by hybridization on pRS-3 labeled with <3> <2> p, as probe. Recombinant viruses were taken and re-titrated on Sf9 cells. Plates of cells without occlusion were identified by visual examination. They were purified three more times and used to prepare virus stocks for further studies.


 Expression of LAV / HTLV III env-related proteins in tissue culture cells infected with recombinant baculovirus Ac-env5
 



  Recombinant AcNPV, carrying the chimeric LAV / HTLV III env gene, has been shown to be able to express proteins related to LAV / HTLV III env by infection of cells in tissue culture. These proteins have been found to be immunoreactive with serum from AIDS patients as well as with monoclonal antibodies which define gp110 and gp41 glycoproteins related to the LAV / HTLV III envelope.



  We infected Sf9 cells (1 x 10 <7>) seeded on a 100 mm dish, with wild-type AcNPV, or its recombinant Ac-env5, at an infection index of 5. 28 hours after infection, the medium was replaced by medium without methionine containing no additional sera, and incubated for 30 minutes. After this period, the medium was replaced by 2 ml of a methionine-free medium containing 100 mu Ci / ml of [ <3> <5> S] methionine, and the labeling was allowed to continue for 2 hours at 28 DEG C.

   At the end of the labeling period, the cells were washed twice with phosphate buffered saline and resuspended in 1 ml lysis buffer, as described above for identification proteins related to the LAV / HTLV III envelope, expressed in cells infected with recombinant vaccinia virus, using immunoprecipitation techniques). Immunoprecipitation was performed as described in the above paragraphs, using serum from AIDS patients as well as mouse monoclonal antibodies against gp110 or gp41.



  The results of this analysis, as presented in FIG. 23, indicate that the recombinant virus Ac-env5 synthesized mainly a protein of 150 kD, related to the envelope of LAV / HTLV III. Since it comigrates with gp150 synthesized by the vaccinia recombinant v-env5 and has been immunoreactive with serum from an AIDS patient as well as with monoclonal antibodies against gp110 and gp41, this protein represents the glycosylated precursor of envelope protein gp150. The transformation treatment of this molecule was not effective, since no gp110 or gp41 were detected during the 2 hour labeling period. However, since the major epitopes of gp110 and gp41 are present on the precursor, this protein is potentially of interest both as a diagnostic reagent and as an immunogen.


 DEPOSIT OF MICRO-ORGANISMS
 



  The following strains of E. coli, carrying the plasmids listed, were deposited in the service called "Agricultural Research Culture Collection (NRRL)", 1815 North University Street, Peoria, Illinois 61 604, (United States of America) and have received the following registration numbers:
 <tb> <TABLE> Columns = 5
 <tb> Head Col 01 AL = L: E. coli
 <tb> Head Col 02 AL = L: Strain
 <tb> Head Col 03 AL = L: Plasmid
 <tb> Head Col 04 AL = L: Registration number
 <tb> Head Col 05 AL = L:

  deposit date
 <tb> <SEP> K12 <SEP> MC1000 <SEP> pv-env1 <SEP> NRRL B-18003 <SEP> 19. 09. 85
 <tb> <SEP> K12 <SEP> MC1000 <SEP> pv-env2 <SEP> NRRL b-18004 <SEP> 19. 09. 85
 <tb> <SEP> K12 <SEP> MC1000 <SEP> pv-env5 <SEP> NRRL B-18005 <SEP> 19. 09. 85
 <tb> <SEP> K12 <SEP> MC1000 <SEP> pv-env7 <SEP> NRRL B-18110 <SEP> 05. 09. 86
 <tb> <SEP> K12 <SEP> HB101 <SEP> pv-gag1 <SEP> NRRL B-18111 <SEP> 05. 09. 86
 <tb> <SEP> K12 <SEP> HB101 <SEP> pAc-gag1 <SEP> NRRL B-18105 <SEP> 29. 08. 86
 <tb> <SEP> K12 <SEP> NF1829 <SEP> pAc-env5 <SEP> NRRL B-18109 <SEP> 05. 09. 86
 <tb> </TABLE>



  The following recombinant viruses have been deposited in the American Type Culture Collection, 12 301 Parklawn Drive, Rockville, MD 20 852, United States of America, and have been assigned the deposit numbers
 <tb> <TABLE> Columns = 3
 <tb> Head Col 01 AL = L: Recombinant virus
 <tb> Head Col 02 AL = L: Deposit number
 <tb> Head Col 03 AL = L: Date of filing
 <tb> <SEP> v-env2 <SEP> ATCC VR 2114 <SEP> 20. 09. 85
 <tb> <SEP> v-env5 <SEP> ATCC VR 2213 <SEP> 20. 09. 85
 <tb> <SEP> v-env7 <SEP> ATCC VR 2148 <SEP> 05. 09. 86
 <tb> <SEP> v-env5NY <SEP> ATCC VR 2149 <SEP> 05. 09. 86
 <tb> <SEP> v-gag1NY <SEP> ATCC VR 2150 <SEP> 05. 09. 86
 <tb> <SEP> Ac-gag1 <SEP> ATCC VR 2147 <SEP> 29. 08. 86
 <tb> <SEP> Ac-env5 <SEP> ATCC VR 2151 <SEP> 05. 09. 86
 <tb> </TABLE>



   It should not be considered that the scope of the present invention is limited only to microorganisms and viruses deposited, since what is deposited constitutes a simple illustration of an aspect of the invention and that all microorganisms or viruses functionally equivalents are within the scope of the present invention. It goes without saying that, without departing from the scope of the invention, numerous modifications can be made to the recombinant viruses, to their chimeric genomes and proteins or peptides and to the vaccine formulations described and represented.



  It also goes without saying that all the dimensions given for the base pairs of the nucleotides constitute approximate information provided mainly for descriptive purposes.


    

Claims (109)

      1. Recombinant virus, characterized in that its genome comprises a nucleotide sequence coding for an envelope epitope or the gag gene of the LAV / HTLV III virus, or for an antigenic portion of the envelope epitope or gag , in which the nucleotide sequence coding for the envelope gene comprises the sequence as shown in FIG. 2, from nucleotide No 5767 to nucleotide No 8549 or any portion of this sequence coding for a peptide or an antigenic protein, or in which the nucleotide sequence of the gag gene comprises the sequence as represented in FIG. 14, from nucleotide No 340 to nucleotide No 1835 or any portion of this sequence coding for a peptide or an antigenic protein,  said nucleotide sequence being under the control of a second nucleotide sequence which regulates the expression of the gene so that a peptide, or a protein related to the LAV / HTLV III epitope is expressed in a host infected with the virus. 2. Recombinant virus according to claim 1, characterized in that the peptide or protein expressed in the infected host is related to an envelope epitope of LAV / HTLV III, or any antigenic portion of this. virus or expression peptide or protein. 3. Recombinant virus according to claim 2, characterized in that the envelope epitope comprises a peptide or a protein having an amino acid sequence as represented in FIG. 2 from the amino acid residue No 1 to the residue 861, or any portion antigen of this sequence. 4. Recombinant virus according to one of claims 1 to 3, characterized in that it comprises a virus comprising an envelope. 5. Recombinant virus according to claim 4, characterized in that it is or comprises a vaccinia virus. 6. Recombinant vaccinia virus v-env5 according to claim 1, such as the ATCC VR 2113 virus or a mutant. 7. Recombinant vaccinia virus v-env2 according to claim 1, such as the ATCC VR 2114 virus or a mutant. 8. Recombinant vaccinia virus v-env7 according to claim 1, such as the virus ATCC VR 2148 or a mutant. 9. Recombinant vaccinia virus v-env5NY according to claim 1, such as the ATCC VR 2149 virus or a mutant. 10. Recombinant virus according to one of claims 1 to 3, characterized in that it is or comprises a baculovirus. 11. Recombinant virus according to claim 10, characterized in that it is or comprises a nuclear polyhedrosis virus. 12. Ac-env5 recombinant baculovirus according to claim 10, such as the ATCC VR 2151 virus or a mutant. 13. Recombinant virus according to one of claims 1 to 3, characterized in that it is or comprises a naked virus. 14. Recombinant virus according to claim 13, characterized in that it is or comprises an adenovirus. 15. Recombinant virus according to one of claims 1 to 3, characterized in that it is or comprises a bacteriophage. 16. Recombinant virus according to claim 1, characterized in that the peptide or protein expressed in the infected host is related to an epitope of LAV / HTLV III core protein or to any antigenic portion of this epitope. 17. Recombinant virus according to claim 16, characterized in that the epitope comprises a peptide or a protein having an amino acid sequence as represented in FIG. 14 of the remainder of amino acids n <o> 1 to 500, or any antigenic portion of this sequence. 18. Recombinant virus according to one of claims 16 and 17, characterized in that it is or comprises an envelope virus. 19. Recombinant virus according to claim 18, characterized in that it is or comprises a vaccinia virus. 20. Recombinant vaccinia virus v-gag1NY according to claim 19, such as the ATCC VR-2150 virus or a mutant. 21. Recombinant virus according to one of claims 16 and 17, characterized in that it is or comprises a baculovirus. 22. Baculovirus according to claim 21, characterized in that it is or comprises a nuclear polyhedrosis virus. 23. A recombinant Baculovirus Ac-gag1 according to claim 22, such as the ATCC VR-2147 virus or a mutant. 24. Recombinant virus according to one of claims 16 and 17, characterized in that it is or comprises a naked virus. 25. Recombinant virus according to claim 24, characterized in that it is or comprises an adenovirus. 26. Recombinant virus according to one of claims 16 and 17, characterized in that it is or comprises a bacteriophage. 27. A process for the preparation of a peptide or an antigenic protein related to an epitope of LAV / HTLV III virus characterized by the implementation, in an appropriate host, of the recombinant virus according to claim 1. 28. The method of claim 27, characterized in that the epitope comprises an envelope glycoprotein of LAV / HTLV III. 29. Method according to claim 28, characterized in that the epitope has an amino acid sequence as described in FIG. 2, from amino acid residue No 1 to residue 861, or any antigenic portion of this sequence. 30. Method according to one of claims 27 to 29, characterized in that the peptide or the protein obtained is purified from the appropriate cultivated host. 31. Method according to claim 27, characterized in that the suitable cultivated host is or comprises a microorganism. 32. Method according to claim 31, characterized in that the microorganism is or comprises a bacterium. 33. Method according to claim 31, characterized in that the microorganism is or comprises a yeast. 34. Method according to claim 27, characterized in that the suitable cultivated host is or comprises a line of animal cells. 35. Process according to Claim 34, characterized in that the peptide or the protein is produced by animal cells infected with the recombinant vaccinia virus v-env5, such as the ATCC VR 2113 virus or a mutant. 36. Method according to claim 34, characterized in that the peptide or the protein is produced by cells infected with the recombinant vaccinia virus v-env2, such as the virus ATCC VR 2114 or a mutant. 37. Method according to claim 34, characterized in that the peptide or the protein is produced by animal cells infected with the recombinant vaccinia virus v-env7, that the virus ATCC VR 2148 or a mutant. 38.  Process according to Claim 34, characterized in that the peptide or the protein is produced by animal cells infected with the recombinant vaccinia virus v-env5NY, such as the ATCC VR 2149 virus or a mutant. 39. The method according to claim 27, characterized in that the suitable cultivated host is or comprises an insect cell line. 40. Method according to claim 39, characterized in that the peptide or the protein is produced by insect cells infected with the recombinant baculovirus Ac-env5, such as the baculovirus ATCC VR 2151 or a mutant. 41. The method of claim 27, characterized in that the epitope comprises a nucleus protein epitope. 42. Process according to claim 41, characterized in that the epitope has an amino acid sequence as shown in FIG. 14 from the remainder of amino acid number 1 to 500 or any antigenic portion of this sequence. 43. Method according to claim 41 or 42, characterized in that the peotide or the protein obtained is purified from the appropriate cultivated host. 44. The method of claim 43, characterized in that the suitable cultivated host is or comprises a microorganism. 45. Method according to claim 44, characterized in that the microorganism is or comprises a bacterium. 46. Method according to claim 44, characterized in that the microorganism is or comprises a yeast. 47. The method of claim 43, characterized in that the suitable cultivated host is or comprises a line of animal cells. 48. Method according to claim 47, characterized in that the peptide or the protein is produced by animal cells infected with the recombinant vaccinia virus v-gag1NY, such as the ATCC VR 2150 virus or a mutant. 49. The method of claim 43, characterized in that the appropriate host is or comprises a line of insect cells. 50. Method according to claim 49, characterized in that the peptide or the protein is produced by insect cells infected with the recombinant baculovirus Ac-gag1, such as the baculovirus ATCC VR 2147 or a mutant. 51. Peptide or protein as obtained by the method according to one of claims 27 to 50. 52. Process for the preparation of an antigenic peptide or protein related to an LAV / HTLV III virus epitope, characterized in that the amino acid sequence of the peptide or protein obtained is determined by means of the method according to one of claims 27 to 50 according to the nucleotide sequence of the chimeric gene contained in the recombinant virus and whether the peptide or protein is synthesized chemically. 53. Peptide or protein as obtained by the process according to claim 52. 54. Formulation of a live virus vaccine, characterized in that it comprises a recombinant virus according to one of claims 1 to 3, this virus being infectious without causing disease for the host to be vaccinated. 55. Formulation of a live virus vaccine according to claim 54, characterized in that the virus is or comprises an envelope virus. 56. A live virus vaccine formulation according to claim 55, characterized in that the envelope virus is or comprises a vaccinia virus. 57. Formulation of a live virus vaccine according to claim 54, characterized in that it comprises an infectious dose of the vaccinia virus v-env5 according to claim 6. 58. Formulation of a live virus vaccine according to claim 54 characterized in that it comprises an infectious dose of the vaccinia virus v-env2 of claim 7. 59. Formulation of a live virus vaccine according to claim 54, characterized in that it comprises an infectious dose of the vaccinia virus v-env7 according to claim 8. 60. Formulation of a live virus vaccine according to claim 54, characterized in that it comprises an infectious dose of the vaccinia virus v-env5NY according to claim 9. 61. A live virus vaccine formulation according to claim 54, characterized in that the virus is or comprises a naked virus. 62. Formulation of a live virus vaccine according to claim 61, characterized in that the naked virus is or comprises an adenovirus. 63. Process for the preparation of a live virus vaccine formulation according to claim 54 from a recombinant virus according to one of claims 1 to 3. 64. Multivalent live virus vaccine formulation, characterized in that it comprises a recombinant virus according to one of claims 2 or 3 and a recombinant virus according to claim 16 or 17, the two recombinant viruses being infectious without causing disease for the host to be vaccinated. 65. A multivalent vaccine formulation according to claim 64, characterized in that each recombinant virus comprises an envelope virus. 66. A multivalent live virus vaccine formulation according to claim 64, characterized in that each enveloped virus is or comprises a vaccinia virus. 67. Multivalent vaccine formulation according to claim 64, characterized in that it comprises an infectious dose of:      (a) vaccinia virus v-env5 according to claim 6, v-env2 according to claim 7, v-env7 according to claim 8 or v-env5NY according to claim 9; and    (b) the vaccinia virus v-gag1NY according to claim 20.   68. Process for the preparation of a mutant vaccine formulation with live virus according to claim 64 from a recombinant virus according to one of claims 2 or 3 and a recombinant virus according to claim 16 or 17. 69. Inactivated virus vaccine formulation, characterized in that it comprises an effective dose of the recombinant virus according to one of claims 1 to 3, in a non-infectious state, in mixture with a pharmaceutical excipient. 70. Inactivated virus vaccine formulation according to claim 69, characterized in that it comprises an effective dose of the recombinant envelope virus according to claim 4, in a non-infectious state, in mixture with a pharmaceutical excipient. 71. Inactivated virus vaccine formulation according to claim 69, characterized in that it comprises an effective dose of the vaccinia virus of claim 5, in a non-infectious state, in mixture with a pharmaceutical excipient. 72. Inactivated virus vaccine formulation according to claim 69, characterized in that it comprises an effective dose of the vaccinia virus v-env5 according to claim 6, in a non-infectious state, in mixture with a pharmaceutical excipient. 73. Formulation of an inactivated virus vaccine according to claim 69, characterized in that it comprises an effective dose of the vaccinia virus v-env2 according to claim 7 in a non-infectious state, in mixture with a pharmaceutical excipient. 74. Formulation of an inactivated virus vaccine according to claim 69, characterized in that it comprises an effective dose of the vaccinia virus v-env7 according to claim 8, in a non-infectious state, mixed with a pharmaceutical excipient. 75.  Inactivated virus vaccine formulation according to claim 69, characterized in that it comprises an effective dose of the vaccinia virus v-env5NY according to claim 9, in a non-infectious state, in mixture with a pharmaceutical excipient. 76. Multivalent inactivated virus vaccine formulation, characterized in that it comprises an effective dose:      (a) the recombinant virus according to one of claims 2 or 3, which expresses the epitope related to the envelope of LAV / HTLV III; and    (b) the recombinant virus according to claim 16 or 17, which expresses the epitope, related to the nucleus of LAV / HTLV III, each recombinant virus being in a non-infectious state, in admixture with a pharmaceutical excipient.   77. A multivalent vaccine formulation with inactivated viruses according to claim 76, characterized in that each recombinant virus is or comprises an envelope virus. 78. Multivalent vaccine formulation with inactivated viruses according to claim 77, characterized in that each envelope virus is or comprises a vaccinia virus. 79. A multivalent vaccine formulation with inactivated viruses according to claim 76, characterized in that it comprises an effective dose of:      (a) the vaccinia virus v-env5 of claim 6, v-env2 of claim 7, or v-env7 of claim 8, or v-env5NY of claim 9; and    (b) the vaccinia v-gag1NY virus of claim 20.   80. A vaccine formulation with subunits, characterized in that the immunogen comprises an effective dose of the peptide or the protein according to claim 51, in admixture with a pharmaceutical excipient. 81. Multivalent subunit vaccine formulation, characterized in that it comprises an effective dose of:      (a) a first peptide or a first protein according to claim 51; and    (b) a second peptide or a second protein according to claim 51, in admixture with a pharmaceutical excipient.   82. Recombinant DNA vector for the production of a recombinant virus according to claim 1, characterized in that it comprises pv-env1 NRRL B-18 003. 83. Single-cell organism, characterized in that it comprises the recombinant DNA vector according to claim 82. 84. Organism according to claim 83 in the form of bacteria, characterized in that it comprises the recombinant DNA vector according to claim 82. 85. Organism according to claim 84, characterized in that it comprises Escherichia coli NRRL B-18 003 or a mutant. 86. Recombinant DNA vector for the production of a recombinant virus according to claim 7, characterized in that it comprises pv-env2 NRRL B-18 004. 87. Unicellular organism characterized in that it comprises the recombinant DNA vector according to claim 86. 88. Organism according to claim 87 in the form of a bacterium, characterized in that it comprises the recombinant DNA vector according to claim 86. 89. Organism according to claim 88, characterized in that it comprises Escherichia coli NRRL B-18 004 or a mutant. 90. Recombinant DNA vector for the production of a recombinant virus according to claim 6, characterized in that it comprises pv-env5 NRRL B-18 005. 91. Unicellular organism characterized in that it comprises the recombinant DNA vector according to claim 90. 92. Organism according to claim 91 in the form of bacteria, characterized in that it comprises the recombinant DNA vector according to claim 90. 93. Organism according to claim 92, characterized in that it comprises Escherichia coli NRRL B-18 005 or a mutant. 94. Recombinant DNA vector for the production of the recombinant virus according to claim 8, characterized in that it comprises pv-env7 NRRL B-18 110. 95. Unicellular organism, characterized in that it comprises the recombinant DNA vector according to claim 94. 96. Organism according to claim 95 in the form of bacteria, characterized in that it comprises the recombinant DNA vector according to claim 94. 97. Organism according to claim 96, characterized in that it comprises Escherichia coli NRRL B-18 110 or a mutant. 98. Recombinant DNA vector for the production of the recombinant virus according to claim 20, characterized in that it comprises pv-gag1NY NRRL B-18 111. 99. Unicellular organism, characterized in that it comprises the recombinant DNA vector according to claim 98. 100. Organism according to claim 99 in the form of bacteria, characterized in that it comprises the vector according to claim 98. 101. Organism according to claim 100, characterized in that it comprises Escherichia coli NRRL B-18 111 or a mutant. 102. Recombinant DNA vector for the production of the recombinant virus according to claim 23, characterized in that it comprises pAc-gag1 NRRL B-18 105. 103. Unicellular organism characterized in that it comprises the recombinant DNA vector according to claim 102. 104.  Organism according to claim 103 in the form of bacteria, characterized in that it comprises the recombinant DNA vector according to claim 102. 105. Organism according to claim 104, characterized in that it comprises Escherichia coli NRRL B-18 105 or a mutant. 106. Recombinant DNA vector for the production of the recombinant virus according to claim 12, characterized in that it comprises pAc-env5 NRRL B-18 109. 107. Single-cell organism, characterized in that it comprises the recombinant DNA vector according to claim 106. 108. Organism according to claim 107, in the form of a bacterium, characterized in that it comprises the recombinant DNA vector according to claim 106. 109. Organism according to claim 108, characterized in that it comprises Escherichia coli NRRL B-18 109 or a mutant.