FR2587720A2 - VACCINE AGAINST ACQUIRED IMMUNODEFICIENT SYNDROME AND IMMUNOGENIC INTERMEDIATES FOR THE PREPARATION OF THIS VACCINE AND IMMUNOLOGICAL ASSAYS FOR THE DETECTION OF THIS SYNDROME - Google Patents

Abstract

ACQUIRED AND INTERMEDIATE IMMUNODEFICITARY SYNDROME VACCINE FOR THE PREPARATION OF THIS VACCINE. THE INVENTION CONCERNS RECOMBINANT VIRUSES WHICH DIRECT THE EXPRESSION OF PEPTIDES OR PROTEINS RELATED TO EPITOPES OF THE ADENOPATHY VIRUS (LAV) AND T-HUMAN LYMPHOCYTE LEUKEMIA (HTLV III) VIRUS, RESPONSIBLE FOR THE AGENT ACQUIRED IMMUNODEFICITARY SYNDROME (AIDS). THESE VIRUSES MAY BE FORMULATED AS LIVE OR INACTIVE VIRUS VACCINES OR MULTIVALENT VACCINES FOR PROTECTION AGAINST LAVHTLV III INFECTION. PEPTIDES AND PROTEINS RELATED TO EPITOPES OF THIS VIRUS, AND THEIR PREPARATION, ARE ALSO DESCRIBED. APPLICATION: VACCINES AND SYNDROME TESTING ASSAYS.

Description

The present invention relates to improvements

  and supplements made to the preparation, described in the

  vaccine, acquired vaccines and, also, the various intermediates used to obtain them. Thus, the present invention relates to viruses which express peptides and proteins related to L-associated lymphadenopathy associated virus (LAV) virus epitopes / human T-cell leukemia virus.

  [Human T-Cell Leukemia Virus (HTLV-III)), the etiologi-

  only Lymphadenopathy Syndrome (LAS) 2 and Acquired Immunodeficiency Syndrome (AIDS); Acquired Immune Deficiency Syndrome (AIDS) J. Viruses

  of the present invention which express peptides

  to LAV / HTLV-III inducing an immuno-protective response

  may be used as immunogens in virus-based vaccine 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-based vaccine formulations.

  live viruses that provide immunogenic stimulation

  and can give rise to strong immunity.

  The present invention also relates to peptides

  and proteins related to epitopes of LAV / HTLV III,

  as immunogens in vaccine formulations - subunits for LAS or AIDS, or in multivalent vaccine formulations, or as antigens in immunoassays for the diagnosis of LAS or AIDS. These pestides and proteins can be produced using

  techniques using recombinant DNA in any

  which vector-host system, or they can be synthesized by chemical methods. Thus, the invention is also directed to the construction of novel DNA and vector sequences comprising plasmid DNA, and viral DNA such as viruses infecting humans, viruses infecting animals, viruses infecting insects. or bacteriophages that can be used to direct the expression of LVA / HTLV-related peptides and proteins in cells of a suitable host from which peptides and proteins can be purified. Chemical methods for the synthesis of peptides and proteins related to LAV / HTLV III are also described. In a specific form of implementation of the

  the present invention, vaccinia viruses have been

  binders to produce proteins related to the envelope or core (core) of LAV / HTLV III. For this purpose, envelope or gaq DNA sequences coding for the glycoproteins of the envelope have been inserted.

  or the core structure proteins of LAV / HTLV III,

  in vaccinia vectors capable of directing

  the expression of the LAV / HTLV III genes in a suitable host.

  The LAV / HTLV III envelope-related proteins, oroduced by recombinant vaccinia viruses, have been found to be antigenic and immunogenic and capable of eliciting humoral and cell-mediated immunity in subhuman primates. Gag-related proteins of LAV / HTLV III,

  produced by the recombinant vaccinia virus, are

  immunoreactive proteins that contain major epitopes

  authentic core proteins.

  Recombinant vaccinia viruses, which express

  proteins related to the envelope of LAV / HTLV III, may

  can be used alone or in concert with vaccinia recombinants that express other LAV / HTLV III related proteins (such as core structure proteins) in virus-based vaccine formulations. Alternatively, LAV / HTL7 III related proteins, produced by recombinant viruses, can be purified or chemically synthesized, and used as immunogens in subunit vaccine formulations). Since the LAV / HTLV III-related glycoprotein (s) may be recognized as "foreign" in the animal host, a humoral and / or cell-mediated immune response will be elicited against that protein or combination of proteins. In an appropriately prepared vaccine formulation, this must protect the host against infections

subsequent Dar LAV / HTLV III.

  In another specific embodiment of the present invention,

  In this invention, recombinant bacculoviruses (Autographa Californica, nuclear polyhedrosis virus, or ANCPV) have been used to produce LAV / HTLV III-related envelope and core proteins. For this purpose, envelope or gag DNA sequences, which encode envelope or core structural proteins, respectively, have been inserted into bacculovirus-type vectors which are capable of directing the expression of the LAV / HTLV III in a suitable host. It has been found that the proteins of LAV / HTLV III,

  produced by recombinant bacculoviruses, are antigenically

  according to the measurement made by radioimmunoprecipitation

and ELISA.

  The present invention also provides for the production of

  antigens of LAV / HTLV III, 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 assays

  Immunoassay and ELISA (Enzyme linked immuno-

  sorbent assay) and radioimmune assays that are useful as diagnostic tools for the detection of LAV / HTLV specific antibodies in blood samples, body fluids, tissues, etc. In addition,

  this reagent will be an interesting tool to elucidate the

  pathogenesis of LAV / HTLV III.

  AIDS (Acquired Immunodeficiency Syndrome) is a disease characterized by severe immune deficiency mainly due to disorders of the patient's cell-mediated immune response (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

  Adenopathy Lymphadenopathy Syndrome (LAS) 7,

  chronic lymph nodes, leukopenia and a quantitative decrease in peripheral blood helper cells (OKT4 cells), leading to a reversal of

  normal ratio of helper lymphocytes to lymphocytes

  T suppressor cells (OKT4: OKT8), which moves from 2 to 0.1 or less as the disease progresses; and (b) an immunodeficiency state characterized by a decrease in OKT4 cells and reversal of 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.

  Some 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 and information

  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 that the agent causing AIDS is a

  retrovirus with a tropism directing it towards the

  helper T cells. These groups are: (a) R.C. Gallo and colleagues at the National Institute of Health have been able to isolate a cytopathic retrovirus

  (HTLV III) on patients with AIDS and pre-existing

  AIDS (R.C. Gallo et al., 1984, Science 224: 500, M. Popovic et al., 1984 (Science 224: 497).) They also detected antibodies against HTLV III in the serum of patients with AIDS.

of AIDS.

  (b) L. Montagnier and his collaborators isolated at the Institut Pasteur a lymphotropic retrovirus (LAV / HTLV III) - - - on a patient - who had cervical lymphadenopathy and also presented a risk of AIDS (Barre -Sinoussi, F. et al., 1983, Science 220: 868). This group could also show the existence of antibodies against LAV / HTLV III in serum from AIDS patients (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 developed AIDS after receiving blood from a donor with AIDS.

  (M. M. Feorino et al., 1984, Science 225: 69).

  (c) J. Levy and colleagues isolated infectious retroviruses (so-called AIDS-associated retroviruses (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 probably all belong to the same subgroup of retroviruses (J. A. Levy et al., 1984, Science 225: 840).

  and they will collectively be called LAV / HTLV III here.

  The general structure of retroviruses is that of a

  nucleus of ribonucleoproteins surrounded by an envelope containing

  lipid that the virus acquires in the course of the bud

  cell. Virally encoded glycoproteins are

  buried in the envelope and protrude outwards.

  laughing. They determine the viruses known to the host and

  lie with specific receptors on the surface

  sensitive cells. Neutral antibodies are thought to be

  lisients are fixed on the glycoproteins of the envelope and block the interaction with receptors located at the

  cell surface (pages 534-535 in "The Molecular Bio-

  Logy of Tumor Viruses, published by John Tooze, 1973, Cold Spring Harbor Laboratory, pages 226-277 and 236237 in "RNA Tumor Viruses", published 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 T4 antigen T-cell cluster, is the receptor or component of the virus receptor (Dalgleish AG et al., 1984, Nature 312: 763, D. Kltazmann et al., 1984, Nature

312: 767).

  The RNA genome of LAV / HTLV III is schematized in Figure 1. Three genes are generally recognized: the gag gene codes for the internal structural proteins (core proteins) of the virus and defines the specific antigens of Zj 7720

  group of viruses. The pol code annoyance for transcriptase

  verse associated with the virion. The discomfort env codes for glycoproteins

  viral teins. Other regions, labeled sor and 3'-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 a response

  active immune system, treatment with chemotherapy agents

  therapy and treatment with interferon.

  Traditional ways of preparing vaccines

  include the use of inactivated or attenuated viruses.

  Mitigation refers to the production of virus strains that have essentially lost their ability to cause disease. One way to achieve this is to subject the virus to unusual growth conditions and / or frequent passage into cell culture. Virus mutants that have lost virulence are then selected

  but are still able to trigger an immune response

  silent. 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 vaccines

  alive, the most troublesome of which is insufficient attenuation.

  Instead of the above methods, so-called subunit vaccines or vaccine subunits can be used. This involves immunization obtained only with the help of proteins containing the immunological material

  concerned. For many enveloped viruses, glycoprotein

  viral-encoded protein contains epitopes capable of

  expensive production of neutralizing antibodies. An advantage of

  subunit vaccines is that the non-intact viral material

ressant is excluded.

  Vaccines are often given in combination with various adjuvants. Adjuvants help to achieve a higher and higher level of immunity, using lower levels of antigen and lower

  doses only if the immunogen was administered alone.

  The use of recombinant DNA technology

  for the production of subunit vaccines implies

  molecular cloning and expression in an appropriate vector

  asked for the viral genetic information coding for

  which can trigger a neutralizing response in the host animal. All other genetic information of 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 entire virus and

  he is not likely to become infected.

  Recently, an approach has been described, which is

  potentially useful for the production of sub-

  units (Mackett, G. L. Smith and B. Moss, 1982, Proc Nat'l.

  Acad. Sci. 79, 7415-7419; M. Mackett, G. L. Smith and B. Moss, 1984, J. Virol. 49, 857-864; D. Ranicali and E. Paoletti, 1982, Proc. Nat'l. 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 virus of

  recombinant vaccinia expresses the inserted foreign gene and triggers

  thus, an immune response of the host to such genes.

  Since recombinant vaccinia viral virus can be used as a vaccine, such an approach combines the advantage of vaccines

  sub-unit (s) and live vaccines.

  The vaccinia virus contains a DNA genome

  (deoxyribonucleic acid) linear double-strand

  about 167 kilobases of bases and it replicates within the cytoplasm of infected cells. These viruses contain a complete system of transcription enzymes (this includes

  styling, methylation and polyadenylation enzymes

  within the core of the virus, which are necessary for

  the infectivity of the virus. The regulatory sequences of trans-

  description of the vaccinia virus (promoters)

  the initiation of transcription by the vaccine RNA polymerase

  but not by the RNA polymerase of the host cells. The expression of foreign DNA in recombinant vaccinia viruses requires the ligation of vaccinia promoters on DNA sequences encoding foreign gene proteins. Plasmid vectors, also called insertion vectors, have been constructed to insert chimeric genes into vaccinia viruses. A type of vector

  of insertion is composed of: (a) a vacant virus promoter;

  including the transcription initiation site;

  (b) several endonuclease cloning sites with

  unique triction, located downstream of the start site of trans-

  cription for the insertion of foreign DNA fragments; (c) non-essential DNA from vaccinia virus (such as the gene

  TK) surrounding the promoter and cloning sites that

  the insertion of the chimeric gene into the non-essential homologous region of the virus genome; and (d) a marker of bacterial origin for replication and resistance to

  antibiotics for replication and selection in E. coli.

  Examples of such vectors are described by Mackett

  (M. Mackett, J. L. Smith and B. Moss, 1984, J. Virol., 49, 857-

  864). Recombinant vaccinia viruses are produced

  by transfection of plasmids for insertion of bacteria recom-

  binaries, containing the foreign gene, to previous cells

  have been infected with the vaccinia virus. A recombinant

  its counterpart 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 plaque hybridization, or genetic selection of recombinant viruses that can be isolated later. These vaccinia recombinants retain

  their essential functions and infectivity, and they can

  can be constructed to have approximately 35 kilo-

foreign DNA bases.

  The expression of foreign genes can be detected by enzymatic or immunological assays (for example by immunoprecipitation, radioimmunoassay or immunoagagulation). Natural membrane glycoproteins

  produced by cells infected with vaccinia recom-

  binante are glycosylated and can be transported to the cell surface. High levels of expression can be achieved by using strong promoters or by obtaining

  by cloning multiple copies of a single gene.

  Recently, an approach has been described which is potentially

  It is particularly useful for the production of recombinant proteins (Pennock et al., 1984, Mol., Cell Biol., 4: 399, Smith et al., 1983, J. Virol., 46: 584). This approach involves the use of a baculovirus vector to express foreign genes inserted into its genome. After introduction into a cell of a suitable host, the recombinant baculovirus

expresses the foreign gene.

  The prototype of the baculovirus family is Auto-

  grapha californica, nuclear polyhedrosis virus (AcNPV).

  The AcNPV genome consists of a double-stranded circular DNA species of 128 kilobase pairs,

  the map with respect to several restriction sites (G.E.

  Smith and M. D. Summers, 1978, Virology 89: 517). The virus is

  replicate in the nucleus of infected insect cells.

  Two forms of virus are produced as a result of infection,

  wild-type AcNPV, sensitive cells,

  occlusion and no occlusion. Occlusion virus particles are surrounded by a protein called polyhedron encoded by the polyhedrin gene (see Virol, 131: 561-565, 1983). In infected cells,

  occlusion viral particles can be easily visualized

  using an optical microscope.

  Expression of foreign DNA in recombinant baculoviruses requires ligation of baculovirus promoters on proteins encoding DNA sequences of the gene

  foreign. Vectors of the plasmid type, also called vectors

  insertion probes, were constructed to insert chimeric genes into AcNPV. One type of insertion vector is composed of: (a) an AcNPV promoter comprising the transcription initiation site; (b) a plurality of single-restriction endonuclease cloning sites located downstream of the transcription start site for insertion of foreign DNA fragments; (c) non-essential AcNPV DNA (such as the polyhedrin gene) flanking the promoter and the cloning sites that direct the insertion of the chimeric gene into the non-essential 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

  transfection of cells with bacterial insertion recom-

  of plasmids containing the gene, with the baculo-

  virus. Homologous recombination occurs within infected cells and causes insertion of the foreign gene into the viral genome. Once a foreign gene has been inserted into the polyhedrin gene, the occlusion virus particles can no longer be produced and the resulting recombinant plaques can be examined visually for evidence.

  the absence of occlusion bodies. Infected cells can

  can also be detected using immunoassay techniques

  logic, hybridization of DNA plaques, or genetic selection giving recombinant viruses that are

  can isolate afterwards. These recombinant baculoviruses

  serve their essential functions and infectivity.

  Expression of foreign genes can be detected by enzymatic or immunological assays (e.g.

  by immunoprecipitation, radioimmunoassay or immune

  nocoagulation). High levels of expression can be achieved

  using strong promoters or by cloning

multiple copies of a single gene.

  Viruses that direct the expression of peptides or proteins related to LAV / HTLV III epitopes will be described. The recombinant viruses of the present invention,

  expressing epitopes related to LAV / HTLV III

  both an immunoprotective response, can be formulated as vaccines containing viruses to protect humans against infection due to LAV / HTLV III. In a particular embodiment of the present invention, vaccine formulations can be prepared for

  viruses, using infectious viruses that express

  such LAV / HTLV III-related epitopes in an infected host, but do not cause such disease in this host. The invention also relates to peptides or proteins apparent to epitopes of LAV / HTLV III. We can formulate

  peptides or proteins that elicit an immune response

  protection, in subunit vaccines or in vaccines

  multivalents to protect humans against infection

  by LAV / HTLV III. Alternatively, the peptides or proteins of the invention may be used for diagnostic assays and assays 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 therefrom; 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 of

  yeast transformed with recombinant plasmids.

  The present invention also provides methods, procedures and DNA constructs for

  the expression of the genetic information coding for epi-

  top of LAV / HTLV III. Alternatively, it is possible to perform the syn-

  chemical thesis of the peptides and proteins of the present invention. In specific embodiments of the present invention, plasmids are inserted into the plasmids.

  gene sequences encoding the enzymatic glycoproteins

  loppe or LAV / HTLV III core structure proteins (i.e. env or gag gene sequences of LAV / HTLV III, respectively), so that an earlier vaccinia virus promoter is placed in the region. 5 'to the starting methionine sequence (ATG) of the gene sequences

  LAV / HTLV III, which gives rise to the construction of chimeric genes

  res flanked or surrounded by thymidine

  kinase (TK) of vaccinia DNA. These plasmids were trans-

  in cells that have been previously infected with wild-type vaccinia virus, thus leaving the env gene or LAV / HTLV III chimeric gag, flanked by the TK sequences, to recombine in the TK gene of the vaccinated. The cells are allowed to lyse and the resulting viruses are cultured on TK- cell plates. The recombinant viruses are selected 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.

  DNA on a suitable radiolabeled probe comprising

  envelope sequences of LAV / HTLV III or gag LAV / HTLV III.

  Positive plaques for the hybridization were purified,

  resulted in the expansion and the viruses were recom-

  resulting binders to assays for their ability to produce LAV / HTLV III envelope glycoproteins or LAV / HTLV III core structure proteins. Proteins related to the envelope of LAV / HTLV III,

  expressed by recombinant vaccinia viruses have been shown to

  are antigenic and immunogenic and capable of

  ter in subhuman primates immunity mediated both humoral and cellular. The gag-related proteins of LAV / HTLV III, expressed by recombinant vaccinia viruses, were immunoreactive proteins that contained the major epitopes of the authentic core proteins. In other specific embodiments of the present invention, env or gag sequences of LAV / HTLV III have been inserted into a plasmid such that a baculovirus polyedrine promoter is placed at the 5 'position relative to the sequence. initiation methionine (ATG) of the LAV / HTLV III gene followed by polyhedrin DNA sequences at the 3 'end. These plasmids were then co-transfected.

  in cells with baculovirus DNA of the wild type.

  ge, leaving the chimeric LAV / HTLV III gene flanked by additional sequences of AcNPV recombine in

  the baculovirus polyhedrin gene. The cells were left

  lysed them and the resulting viruses grown on plates. By visual examination to detect the absence of occlusion bodies or by DNA-DNA hybridization on radiolabeled env / HTLV III or gag probes of LAV / HTLV III,

  the selection of recombinant viruses was carried out. We have puri-

  releasing the recombinant plaques and provoked

  and the resulting recombinant viruses were screened for their ability to produce

  proteins related to LAV / HTLV III by immunoprecipitation

  followed by analysis on SDS (sodium dodecyl sulphate) polyacrylamide gels. Lysates of infected cells were subjected to assays for their ability to

  detect antibodies to LAV / HTLV III in serum by ELISA.

  The accompanying figures will now be briefly described.

  Figure 1 shows the integrated provisional genome structure of LAV / HTLV III. The hatched areas indicate regions of

  frames' open reading. The coding regions

  for the group-specific antigen, the reverse transcriptase

  se and the envelope proteins are designated respectively by gag (group-specific antigen), pol and env. The overlapping open reading frames are represented by

  crosshatch areas. Numbers are the names

  pairs of base pairs downstream of the styling site ("cap" site), where viral transcription starts. The restriction sites are labeled as follows: Bg, BglII; Ec, EcoRI;

Hn, HindIII; kp, kpnI; Ss, SstI.

  FIGS. 2A and 2B show the nucleotide sequence of the LAV / HTLV III specific region (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-env1, pv-env2 and pv-env5 are indicated. The entire amino acid sequence of the envelope gene, as deduced from the nucleotide sequence data, is

also indicated.

  Figure 3 is a schematic representation of plasmid construction containing a portion of the sequence encoding LAV / HTLV III envelope proteins, sequence inserted downstream of a vaccinia virus promoter. The coding sequence for 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 / HTL III envelope is shown at the bottom of the figure. Underlined sequences indicate codons

  suspected priming and reading frame for the chimeric gene

  America. It will be noted 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 sequence

  Envelope coding of LAV / HTLV III.

  Figure 4 is a schematic representation of plasmid construction containing the entire LAV / HTLV III envelope protein coding sequence.

  inserted downstream of a vaccinia virus promoter. The promotion

  Vaccinia virus 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 'and 3' portions of the coding region were first cloned separately into pGS20 to form pv-envl which contains the amino terminus of the gene coding.

  the envelope of LAV / HTLV III and pv-env2 which contains the

  the carboxyl coding of the LAV / HTLV III envelope gene. These two portions are grouped together at the StuI site as

represent.

  Figure 5 is a schematic representation of the plasmid construct containing the LAV / HTLV III envelope protein coding sequence, without the transmembrane (anchor) sequence, inserted downstream of a vaccinia virus promoter. Plasmid pv-env7 was constructed in two steps. The 5 'portion of pv-env was inserted into p26 to form an intermediate plasmid, pv-env5 / 26, which provided a translational terminator sequence (TAA) giving a truncated env gene. The truncated env sequence, terminated by TAA, was then inserted into pG20 to construct pv-env7 in which the truncated env gene is flanked.

  by vaccinia TK gene sequences.

  Figure 6 shows the construction and the choice

  recombinant vaccinia virus. The solid bars represent

  feel the thymidine kinase (TK) gene of 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 promoter of the TK gene.

  vaccine (hatched bar) and by the coding region of

  LAV / HTLV III loppe (open bar). After infection of the cells with vaccinia, the recombinant plasmid containing the TK gene interrupted by the LAV / HTLV III envelope gene is introduced into the infected cells. The recombinations that occur in the TK sequences surrounding the chemical gene introduce the LAV / HTLV III envelope gene sequence into the vaccinia virus genome. The resulting recombinant virus, containing the LAV envelope gene /

HTLV III, is TK.

  FIGS. 7A and 7B show a characterization of the structures

genomes of recombinant vaccinia viruses containing the env gene of a LAV / HTLV III. Figure 7A shows a

  analysis, using a restriction enzyme, recombinants

  v-env5 and v-env5NY vaccines as well as their

  respective parental vaccines (WR and v-NY, respectively).

  DNA fragments generated by digestion products

  by the restriction enzyme HindIII have been resolved by

  trophoresis on an agarose gel and stained with bromide

  of ethidium to visualize the bands. Figure 7B shows

  feels the Southern clot (Southern) obtained after transfer

  fragments resolved by restriction on nitrocellulose

  lose and hybridization on a probe with translation by strangling

  specifically, specific LAV / HTLV III envelope sequences.

  FIG. 8A represents an immunocautery analysis.

  Western ("West") regulation of proteins produced by vaccinia env-LAV / HTLV III recombinants. Proteins from

  following sources were resolved on a polyacrylate gel

  gradient amide of 7 to 15% SDS and electrotransfered on nitrocellulose paper: LAV / HTLV III virion proteins (LAV / HTLV III); cells infected with wild-type vaccinia virus ("WTvv"); ("Wild-type vaccinia

  viruses "), uninfected cells (" mock "), infected cells

  by recombinant viruses v-env2 (v-env2) or v-env5 (v-env5). Immunotherative proteins against the serum of a patient with AIDS were detected by the action

  peroxidase conjugated to anti-human IgG antibodies.

  The LAV / env genes produced are indicated by gp150, gp101 and gp41. The molecular weight standards are expressed in

kilodaltons (kd).

  FIG. 8B represents an analysis of an immunocautery

  Western gulat of proteins produced by LAV-env vaccinia recombinants in two types of cells. Proteins from BSC-40 or HeLa cells were resolved by SDS-polyacrylamide gel electrophoresis

  (SDS-PAGE) and electro-transferred onto nitrocellulose paper

  then reacted with pooled serum from LAV / HTLV-III serum positive persons; lanes 1 and 5 represent cells infected with "mock"; lanes 2 and 6 represent cells infected with 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 125I-labeled Protein A. Products of the gene type

  LAV-env are indicated by gp150, gpl10 and gp41.

  Figure 9A shows the results of radioimmunoprecipitation analysis of proteins synthesized in cells infected with wild-type vaccinia virus ("WTvv") or with recombinant vaccinia virus (v-env2 and v-env5). The proteins were labeled with 35 S-methionine 10 to 12 hours after infection. The labeled proteins present in the cell lysates were reacted with control human serum (N) or with serum

  patient with AIDS (I) and provoked the precipi-

  complex immunization with Staphylococcus aureus protein A. The immunoprecipitated proteins were resolved by electrophoresis on a 15% SDS polyacrylamide gel and detected by fluorography. Molecular weights are

indicated in kilodaltons (kd).

  Figure 9B shows the results of an analysis

  by radioimmunoprecipitation of 3H-labeled proteins

  glucosamine produced by the expression effect of the vacciniaLAV / HTLV III recombinants of the invention. Hela cells were either

  "infected" by non-infectious cells ("mock";

  infection) (lanes 1 and 5), either infected with wild-type vaccinia viruses (lanes 2 and 6), v-env5 (lanes 3

  and 7) or v-env2 (tracks 4 and 8) and marked with 3H-

  glucosamine. The cell lysates were immunoprecipitated with normal human serum (lanes 1-4) or with pooled serum from LAV / HTLV III positive serum (lanes 5-8) and protein A. Immunoprecipitated proteins were resolved by electrophoresis on

SDS-polyacrylamide gel.

  FIG. 9C shows the results of a "pulse-hunting" radioimmunoprecipitation analysis of

  LAV / HTLV III envelope proteins produced by the recom-

  vaccinia binoculars LAV / HTLV III. HeLa cells were infected with wild-type vaccinia virus, v-env5 or v-env2 as indicated, labeled with 35 S-methionine, and washed with flushing medium. At the following intervals of

  The cells were washed, lysed and immunized

  precipitated with pooled serum from LAV / HTLV III serum-positive individuals and resolved by SDS-PAGE (polyacrylamide gel electrophoresis): 0 hours (lanes 1, 7, 13); half an hour (tracks 2, 8, 14); 1 hour (lanes 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 14C-labeled molecular weight standards. Figure 9D shows the results of a radioimmunoprecipitation assay for LAV / HTLV III envelope-related proteins found in cells and in media from cells infected with the cells.

  recombinant vaccinia-LAV / HTLV III virus of the invention.

  HeLa cells were either "infected" with uninfected (i.e. uninfected, lane 1 "mock") viruses or infected with wild-type vaccinia virus (lane 2)

  v-env5 (lane 3) or v-env2 (lane 4) and marked by 35S-

  methionine. The cells were separated from the medium and lysed.

  The cell lysates (pellet) and the supernatant upper medium were each immunoprecipitated with serum group from LAV / HTLV III positive serum individuals. Immunoprecipitated proteins were resolved by

  Polyacrylamide gel electrophoresis (SDS-PAGE).

  Figure 9E shows the result of radioimmunoprecipitation analysis of LAV / HTLV III envelope-related proteins in cells and in

  from cells infected with viruses recom-

  vaccinia-LAV / HTLVL III binders of the invention. Recombinant v-env5 contains the entire coding sequence of the LAV / HTLV III envelope, whereas v-env7 contains the LAV / HTLV III envelope coding sequence except the transmembrane region (anchoring). Hela cells were either "infected" with uninfected (i.e., uninfected, lane 1 "mock") viruses or infected with wild-type vaccinia virus (lane 2), v-env5 (lane 3) or v-env7 (lane 4), and labeled with 35 S-methionine. The cells were separated from the medium and lysed. Cell lysates

  (pellet) and the medium (supernatant) were each immunoprecipitated

  with serum pooled from serum

  positive for LAV / HTLV III. Immunoprecipitation proteins

  cipitates were resolved by electrophoresis on poly-

acrylamide (SDS-PAGE).

  Figure 10 shows an immunocautery analysis.

  Western-type gating of serum samples from mice immunized with vaccinia-LAV / HTLV III recombinant viruses. The mice had been immunized with recombinant vaccinia virus v-env5 or v-env2. After 8 weeks, serum samples were reacted with LAV / HTLV III virion proteins that had been resolved by electrophoresis on SDS-PAGE (polyacrylamid gel) and electrotransferred.

  on nitrocellulose paper. An immunoglobulin was used

  conjugated goat anti-mouse bulin & phosphatasa alkalina to detect LAV / HTLV III proteins that have been recognized by mouse sera. Tracks were serum samples from 5 individual mice inoculated v-anv5, and tracks f through k represent serum samples from 5 individual mice inoculated v-env2. Pooled sera from LAV / HTLV III positive (AIDS) and non-immune C57B16J- (NMS) mice served as controls.

  positive and negative, respectivament. The positions of the glyco-

  LAV / HTLV III envelope proteins gpl50, gpl10 and gp43 are indicated.

  Figure 1i is a histogram representing the steroids

  conversion of male monkeys vaccinated with recombinant vaccinia virus, v-env5. Four macaque monkeys were inoculated, by scarification of the skin, 2 x 10 plaque forming units and 4 monkeys 2 x 107 plaque forming units.

  of v-env-5. One animal was inoculated with 2 x 107 units of

  Recombinant vaccinia-herpes simplex gD (v-HSVgD1) recombinant plaque plate. Ten weeks after the first inoculation, all animals except No. 81 were given a second inoculation of 2 × 10 8 plaque forming units of the same virus. Serum samples were collected prior to inoculation (open bars); 4 weeks after the first inoculation (hatched bars, and 4 weeks after the second

  inoculation (solid bars). We have checked and titrated the sero-

  ELISA conversion, using purified virions of

LAV / HTLV III as target antigen.

  Figure 12 shows an immunocautery analysis.

  Western blot, serum samples from macaque monkeys immunized with recombinant vaccinia-LAV / HTLV III viruses, v-env5, as described for FIG.

  collected virus samples before the first inoculation

  pre-run and 4 weeks after the second inoculation. Serum aliquots were diluted 50-fold and reacted with LAV / HTLV V virion protein which was resolved by polyacrylamide gel chromatography (SDS-PAGE) and immobilized on filters.

  nitrocellulose, by electrotransfer using a proto-

  This method allows the optimal detection of the two envelope glycoproteins, gpl10 (FIG. 12A) and gp41 (FIG. 12B). The LAV / HTLV III proteins recognized by macaque sera were detected by goat anti-human immunoglobulin conjugated to alkaline phosphatase. We used

  as a positive control group sera collected from

  Aru positive for the research of LAV / HTLV III (AIDS). Authentic virions were used as a witness

of LAV / HTLV III (AIDS).

  FIG. 13 represents an immunocautery analysis.

  Western determination of these serum samples from chimpanzees immunized with NY-LAV / HTLV III vaccinia recombinant viruses, v-env5NY. To two chimpanzees, 5 x 108 v-env5NY plaque formation units were inoculated intradermally, while the same dose of v-HSVgD1NY, a vaccinia-herpes simplex virus recombinant, was inoculated into an animal. gD constructed from the same strain of vaccinia

  Parental-NY. All animals received a second inoculation

  8 weeks after the first inoculation. Serum samples were collected prior to immunization (lane 1), 8 weeks after primary immunization (lane 2) and 2 weeks after the second immunization (lane 3). Aliquots of serum were diluted several times and reacted with LAV / HTLV III virion proteins which were

  resolved by polyacrylamide gel electrophoresis (SDS-

  PAGE) and immobilized on nitrocellulose filters by electrotransfer, using a protocol that allows the best possible detection of gp 41. The LAV / HTLV III proteins recognized by the sera of chimpanzees were disappointed.

  by conjugated goat anti-human immunoglobulin

  for alkaline phosphatase. Positive sera were pooled sera from individuals

  serum was positive for LAV / HTLV III (AIDS).

  Figures 14A to 14D show the nucleotide sequence

  specific region of LAV / HTLV III (SstI to KpnI) pre-

  in pKS-5 plasmid DNA; the entire gag LAV / HTLV III gene (nucleotides 340 to 1835) is contained in the inserted portion of LAV / HTLV III. The restriction sites used for the construction of pAc-gagl are indicated. All the amino acid sequences of the gag gene, as inferred from the sequence information

  nucleotides, is also indicated.

  Figure 15 is a schematic representation of the construction of the pAc-gagl plasmid containing the LAV / HTLV III gag protein coding sequence inserted downstream of an AcNPV promoter. The cloning vector pAc610 contains nucleotide sequences corresponding to the polyhedrin gene promoter and comprises 5 'leader sequences and 3' polyhedrin sequences interrupted by sites.

  of cloning located downstream of the transcription start site.

  The cloning sites at position -8 are EcoRI, SstI, SmaI (XmaI), BamHI, XbaI and PstI. SalI, AccI and HincII are not

  not unique. There are no sites for KnpI or BglII.

  Figure 16 schematically shows the construction of

  selection and selection of the AcNPV (Ac-gag1) recombinant. The hatched bar indicates the 5 'polyhedrin and leader promoter sequences. The solid bars represent the baculovirus polyhedrin gene. Portions of this gene are present in the plasmid pAc-gagl DNA, interrupted by the coding region of LAV / HTLV III gag (open bar). of the

  cells are co-transfected with plasmid DNA recom-

  with portions of the interrupted polyhedrin gene

  stained by LAV / HTLV III gag gene (pAc-gagl) with DNA

  wild-type AcNPV. Recombinations that occur

  in the polyhedrin sequences flanking the chimeric gene introduce the LAV / HTLV III gag gene sequence.

in the genome of AcNPV.

  FIG. 17 represents a coagulation analysis of Northern (Northern) RNA extracted from Spodoptera frugiperda cells infected with wild-type AcPNV and Ac-gagl,

  using specific waves for LAV / HTLV III gag.

  Figure 18 shows the result of a radioimmunoprecipitation analysis of the proteins expressed in

  cells infected with recombinant baculovirus Ac-gagl.

  Proteins were labeled with (35S) methionine for 2 hours at 24 hours (lanes 1, 2), 48 hours (lanes 3, 4)

  and 72 hours (lanes 5, 6) after infection. We made

  the labeled proteins present in the cell lysates with control human serum (lanes 1, 3, 5) or with the serum of a patient with AIDS (lanes 2, 4, 6), and the immun-complexes were precipitated by protein A of Staphylococcus aureus. The immunoprecipitated proteins were resolved by electrophoresis on a polyacrylamide gel

  at 10% SDS and detected by fluorography. The molecular weights

  Cells are given in kilodaltons.

  Figure 19 shows the results of a "hunting pulse" radioimmunoprecipitation analysis of the LAV / HTLV III gag-related proteins 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 post-infection. The cells were then washed with complete medium and incubated with complete medium for 0 h (lanes 1, 2), 2 h (lanes 3, 4), 4 h (lanes 5, 6) and 8 h ( tracks 7, 8). At the indicated times, cells were washed, lysed

  and immunoprecipitated with pooled serum from

  seropositive for LAV / HTLV III (lanes 2, 4, 6, 8) or normal human serum (lanes 1, 3, 5, 7). The proteins

  resolved by SDS-polyacryl gel electrophoresis.

lamide.

  Figure 20 schematically shows the construction

  of five plasmids, pv-gag1, pv-gag2, pv-gag3, pv-gag4 and pv-gag5 containing the 7.5K vaccinia promoter fixed by ligation to various lengths of the LAV / HTLV III genome containing sequences gag coding. The cloning vector pGS62 used in these constructions is identical to pGS20 (see FIG.

  single EcoRI site downstream of the unique SmaI site of pGS20.

  Figure 21 shows the results of a type immunocoagulation assay. Western (West) proteins expressed in cells infected with recombinant vaccinia LAV / HTLV III viruses (v-gaglNY, v-gag2NY, v-gag3NY, v-gag4NY and v-gag5NY), the starting vaccinia virus ( NY) and "infected" cells by non-infectious cells ("MOCK"). Samples of purified LAV / HTLV III virions ("LAV / HTLV III") were included as positive controls. Confluent BSC-40 cells were infected at a multiplicity index of infection of 10. The infection was left

  continue for 12 hours, at the end of which we collected

  cells and lysed them. Cell Proteins

  total were resolved by SDS-gel electrophoresis.

  PAGE and immobilized by electrotransfer on nitro-

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

alkaline phosphatase.

  Figure 22 schematically represents the construction of

  Plasmid pAc-env5 containing the entire LAV / HTLV III envelope coding sequence inserted downstream of an AcNPV polyhedrin promoter. The cloning vector pAc610

  is depicted in Figure 15 above.

  Figure 23 shows the results of a radioimmunoprecipitation analysis of the proteins expressed in

  cells infected with recombinant baculovirus Ac-

  env5. "Infected" cells with non-infectious viruses ("MOCK") and the starting baculovirus strain (AcNPV) are included as controls. Proteins expressed by infected Sf9 cells were labeled, 28 hours after infection, for 2 hours with 35 S-methionine. The labeled proteins present in the cell lysates were reacted with AIDS patient serum or with mouse monoclonal antibodies which define gpl10 or gp41, and the immun-complexes were precipitated using protein A of Staphylococcus aureus. It was resolved by SDS-PAGE gel electrophoresis and detected by

  fluorography immunoprecipitated proteins.

  We will now present a description more

detailed description of the invention.

  The present invention aims at obtaining viruses which

  produce the peptides; and proteins related to epi-

  top of LAV / HTLV III. The invention also provides peptides and proteins related to LAV / HTLV III epitopes that can be produced using recombinant DNA or chemical synthesis methods. The viruses or peptides and proteins of the present invention which can

  elicit an immune protection response can serve

  of immunogens in various vaccine formulations, including multi-valent vaccine formulations, to protect against infection with LAV / HTLV III, which is the causative agent of LAS (Adenopathy Syndrome) and

  AIDS. Alternatively, the antigens

  tides or proteins of the invention, in diagnostic immunoassays or assays for detecting, in a patient, antibodies specific for LAV / HTLV III. Peptides

  or proteins that are used in immunoassays or assays.

  of the invention must be antigenic (that is,

  ie, capable of reacting with a patient's antibodies to LAV / HTLV III), but they do not need to be immunogenic (i.e., capable of eliciting a response

  immune). Moreover, it is not necessary for

  genes used in the immunological assay or assay of dia-

  In vivo, immunosuppressive responses provide immunological protection. In accordance with one embodiment of the present invention, recombinant DNA techniques are used to insert nucleotide sequences encoding LAV / HTLV III epitopes into expression vectors that will drive expression. LAV / HTLV III sequences in appropriate host cells. These host cell expression vector systems can be used to produce LAV / HTLV III related peptides and proteins in vitro and, in this case, the produced genes can be purified from cultured cells. Peptides or proteins that can elicit an immune protection response and

  immunogen in subcutaneous vaccine formulations

  units. Alternatively, peptides and proteins can be used.

  ines, immunogens or simply antigenic, of the invention

  as antigens in immunoassay tests and assays.

  designed to detect antibodies from specific patients.

  of LAV / HTLV III. In another approach to the production

  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 recombinants that express peptides and

  antigenic and / or immunogenic tegins related to LAV / HTLV III. These peptides and proteins can then be chemically synthesized and used in synthetic subunit vaccine formulations (if they are immunogenic)

  or as antigens in immunoassay tests or assays

  diagnoses (if they are antigenic and / or

  nogènes). When the expression vector is a virus that directs the expression of an immunogen linked to an epitope of LAV / HTLV III capable of evoking an immune protection response, the virus itself can be formulated as a vaccine. Infectious recombinant viruses, which do not trigger disease in the host, can be used in

  of virus vaccines 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 other

diseases.

  For the sole purpose of presenting a clear description,

  the process of the invention can be divided into the following steps:

  (a) isolation of a gene, or gene fragment, encoding LAV / HTLV III virus proteins; (b) inserting the gene, or gene fragment, into expression vectors; (c) identifying and growing the recombinant expression vector in a host system that is capable of replication and gene expression; (d) identifying and purifying the product obtained with the gene; (e) determination of the immunity of the product, and

(f) formulation of a vaccine.

  In specific embodiments of the invention, the construction of recombinant vaccinia virus and baculovirus containing the envelope gene is described.

  of LAV / HTLV III, which directs the expression of

  logically bound to LAV / HTLV III envelope proteins in tissue culture cells infected with recombinant viruses. In other embodiments of 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 LAV nuclei structural proteins. / HTLV III in culture cells of these tissues infected with recombinant viruses. However, the compositions and methods described herein are not limited to constructing recombinant viruses expressing LAV / HTLV III coat or gag-related proteins, and can be used to construct recombinants in

  any expression vector system for the production of

  polypeptides related to antigens of any

which ethicological agent of AIDS.

  For the sake of clarity, the entire process will be studied in terms of 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, but are not limited to, LAV gene products.

  HTLV III, as gag, pol and env and at least four genes sup-

  are now called sor, tat, 3'-orf and a gene which is variously called art or trs (see Fisher et al, 1986,

Science 233: 655-659).

  ISOLATION OF GENES, OR GENE FRAGMENTS, ENCODING

VIRAL PROTEINS LAV / HTLV III

  The isolation of the LAV / HTLV III genes involves all

  first the isolation of DNA fragments containing the sequences

  these envelope genes. As previously explained, LAV / HTLV III has an RNA genome and, therefore, it is possible to obtain

  the corresponding DNA which encodes the LAV / HTLV III (a) gene

  cDNA RNA isolated from purified virions of LAV / HTLV III, or (b) by cDNA cloning of polylAI-containing RNA obtained from cells infected with LAV / HTLV III, or (c) ) by cloning purified genome DNA from

  of cells infected with LAV / HTLV III. Hereinafter, we call

  the DNA coding the LAV / HTLV III genes of LAV DNA /

HTLV III.

  In order to generate DNA fragments of LAV / HTLV

  III, the LAV / HTLV III DNA can be cleaved at specific sites.

  using various restriction enzymes. In

  Alternatively, DNA can be used in the presence of

  ganesis to fragment DNA, or we can shear

  physically DNA, for example by sonication.

  The linear fragments of DNA can then be separated according to

  their dimensions, by conventional techniques, which

  takes, but is not limited to, electrophoresis on agar and polyacrylamide gel and column chromatography.

  Any restriction enzyme may be used

  or combination of restriction enzymes to generate one or more LAV / HTLV III DNA fragments containing the gag or envelope sequences, provided that the enzymes do not destroy the antigenicity of the protein product of the invention. gene. For example, the antigenic site of a

  Protein can be from about 7 to about 14 amino acids.

  Thus, a protein of the size of the precursor peptide

  envelope (approximately 97 000 daltons) may contain

  several separate antigenic sites, perhaps thousands considering overlapping sequences, secondary and tertiary structural considerations, and treatment possibilities such as acetylation, glycosylation, or phosphorylation. Therefore, many partial sequences of envelope polypeptide genes are likely to encode

  an antigenic site. Therefore, we can use

  many combinations of restriction enzymes to generate

  fragments of DNA which, when inserted into a vec-

  are capable of directing the production of specific envelope amino acid sequences comprising

  different antigenic determinants.

  Once the DNA fragments have been generated, we can

  to identify in a number of ways an identification of the specific DNA fragment containing the LAV / HTLV III envelope gene. Firstly, it is possible to sequence the DNA fragments corresponding to the entire genome of LAV / HTLV III, then to identify the fragments containing the protein sequence of envelope or gag gene genes on the basis of a comparison of the predicted amino acid sequence and the amino acid sequence of the envelope protein

  or gag core proteins. Secondly, once

  mined the entire genome sequence, one can order

  from 5 'to 3' large open reading frames. As the organization

  Genomeization of all retroviruses examined so far is 5'gag-pol-env-3 ', the large open-reading frame closest to the 3' end is very likely to encode the envelope gene. , while the large open reading frame closest to the 5 'end is very likely to encode the gag gene. Thirdly, it is possible to make a probable identification of a specific gene by recognizing the homology with other known retrovirus genes, by nucleic acid hybridization analyzes or by sequence comparisons if the

sequences are known.

  Alternatively, the fragment containing the protein gene

  envelope can be identified by mRNA selection.

  In this procedure, the LAV DNA fragments are used.

  HTLV III to isolate, by hybridization, complementary mRNAs

  tary. Immunoprecipitation analysis of the in vitro translation products of the isolated mRNAs identifies the mRNA and therefore,

  complementary LAV / HTLV III DNA fragments which contain

  the envelope protein sequences. Finally, specific mRNAs of envelope proteins can be selected by

  adsorption of polysomes isolated from infected cells

  by LAV / HTLV III on immobilized antibodies directed

  against envelope or gag proteins. We can synthetize

  radiolabeled envelope DNA, cDNA (complementary DNA

  using the chosen mRNA (from

  adsorbed polysomes). Radiolabeled mRNA or radio-labeled cDNA

  can then be used as a probe to identify LAV / HTLV III DNA fragments containing

  envelope or gag genes. Instead of isolating the enve-

  of work or gag, we can, without this list being limited

  tative, perform the chemical synthesis of the gene sequence itself (provided the sequence is known) or transform cDNA into mRNA that encodes the coat or gag gene. Once identified and isolated, the DNA fragment LAV / HTLV III containing the interesting sequences can be

  first inserted into a cloning vector, such as a sec-

  plasmid cloning, which serves to transform

  Suitable host cells to cause replication of the DNA to generate many copies of the relevant LAV / HTLV III sequences. This can be achieved by ligating the LAV / HTLV III DNA fragment into a cloning vector with complementary coherent termini. However, if complementary restriction sites serve to fragment LAV / HTLV III DNA

  are not present in the cloning vector, the extremes

  mutated DNA molecules may be modified. Such modifications include the production of blunt ends by digestion of the single-stranded DNA termini or by packing the single-ended termini so that the ends can be blunt end ligation. Alternatively, any desired site can be produced by ligating nucleotide sequences (binding agents) on the DNA termini; these ligated binding agents may comprise specific oligonucleotides, synthesized chemically, and coding restriction site recognition sequences. according to

  other processes can be modified by homogeneous termination

  polymer, the cleaved vector and the LAV / HTLV III DNA fragment.

  Transformation of host cells by molecules

  Recombinant DNA molecules that incorporate the isolated gene, by cDNA or a synthetically obtained DNA sequence, make it possible to generate multiple copies of the gene. So, the gene can

  to be obtained in large quantities through the growth of trans-

* Formants, the isolation of 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 surrounded by virus sequences allowing for genetic recombination into virus-infected cells, so that the gene can be inserted into the

viral genome.

  The entire genome of LAV / HTLV III was cloned and sequenced by WainHobson et al. (S. Wain-Hobson et al., 1985, Cell 40: 9). A clone, termed J19 lambda, contained a 9.2 kilobaseous base DNA fragment of an LAV genome sequence inserted into the lambda Hind III site.

L 47.1.

  A particularly useful subclone of lambda J19, containing the LAV / HTLV III envelope gene, is pRS-3 which

  consists of a fragment at 3840 base pairs EcoRI to SstI.

  of the LAV / HTLV III nucleotide sequence inserted into the EcoRi and SstI site of pUC18. The specific LAV / HTLV III DNA 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 nucleotide coding sequences, in the practice of the present invention, for the cloning of the LAV / HTLV III envelope gene, other DNA sequences which encode substantially the same amino acid sequence as shown in FIG. 2. This includes, but is not limited to, nucleotide sequences comprising all or portions of the envelope nucleotide sequence shown on FIG.

  Figure 2, which are amended by replacing

  codons which encode the same amino acid residue or a functionally equivalent amino acid residue within the sequence (for example, an amino acid of the same polarity), producing

a silent change.

  Particularly useful subclones of lambda

  J19 containing the gag gene of LAV / HTLV III are pKS-5 and pSS5.

  The plasmid pKS5 consists of a fragment of LAV / HTLV III com-

  carrying 3148 base pairs of SstI to KpnI in pUC18.

  The specific LAV / HTLV III DNA 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. 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 contains

  nude in pSS-5 comes from the SstI site at nucleotide 224 to the SalI site 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

  the degeneracy of the coding sequences of

  tides, the practice of the present invention can be used,

  for the cloning of the LAV / HTLV III gag gene, other sequences

  those of DNA which encode substantially the same sequence of amino-

  same as that shown in Figure 14. This includes, but is not limited to, nucleotide sequences comprising all or portions of the sequence of

  gag nucleotides 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 a

  same polarity), thus producing a silent change.

  INSERTING ENCODING SEQUENCES OF LAV / HTLV PROTEINS

III IN EXPRESSION VECTORS

  The nucleotide sequence coding for the LAV / HTLV III envelope protein, or part of it, is inserted.

  this sequence, in an appropriate expression vector, is it

  a vector that contains the elements necessary for transcription and translation of the coding sequence

  of inserted proteins. Various host systems can be used

  vectors 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 (eg baculovirus); microorganisms such as yeast containing vectors or yeast bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA. The expression elements of these vectors vary in strength and specificity. According to the host-vector system

  that we use, we can use any of

  a number of suitable transcription and translation elements. For example, when cloning into mammalian cell systems, one can use

  promoters isolated from the genome of mammalian cells.

  fers (for example a mouse metallothioniene promoter) or isolated from viruses which grow in these cells (for example the vaccinia virus 7.5K promoter). Promoters can also be used

  produced by recombinant DNA or by synthetic techniques.

  theories, to ensure the transcription of

  Rees. Specific bootstrapping signals are also required for efficient translation of the inserted protein coding sequences. These signals include the ATG initiation codon and the adjacent sequences. When we insert in the

  appropriate expression vectors the entire enve-

  loppe or gag of LAV / HTLV III, including its own priming codon and the flanking sequences, control signals

  additional requirements for translation may not be necessary.

  sary. However, when inserting only a part of the envelope coding sequence, or gag, it is necessary to provide

  exogenous translational control signals, which

  takes the initiation codon or ATG translation initiation.

  The initiation 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

  of the inserted sequence. These exogenous translational control signals and these codons of iniation or priming of

  translation may have various origins, both natural and

only 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 signals

  requested transcription / translation command and

2587720 '

  protein coding sequences. These processes can

  take techniques using recombinant DNA "in vitro" and synthetic techniques and recombination

  "in vivo" (genetic recombination).

  In the particular embodiments described in detail in the examples of the present invention, vaccinia virus and baculovirus have been chosen as expression vectors. However, the invention does not

  not limit the use of vaccinia virus or

  lovirus. As previously explained, the vectors of expres-

  can not be limited to

  the following vectors or their derivatives: infected viruses

  both human beings and animals such as vaccinia virus or adenoviruses; viruses infecting insects such as baculoviruses; yeast vectors; bacteriophage-like cells, and DNA-like vectors

  plasmid and cosmid, to name 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 adenovirus by recombinant

  its "in vitro" or "in vivo". Insertion into a non-essential region of the virus genome (e.g. E1 or E3 region) will result in a recombinant virus that is viable and capable of expressing, in infected hosts, the related protein.

  to LAV / HTLV III. Currently, there are two strains of adeno-

  viruses (types 4 and 7) approved and used as vaccines for military personnel. They are the first candidates to use as vectors to express genes

of LAV / HTLV III.

  In addition, one can choose a strain of cells

  which modulates the expression of inserted sequences, or modifies

  and process the produced chemical gene in the desired specific manner. The expression of certain promoters can be increased in the presence of certain inducers (for example zinc ions

2587720 '

  and cadmium for metallothionein promoters). Thus, the expression of the genetically engineered LAV / HTLV III protein can be regulated. This is important if the product

  protein from the cloned foreign gene is lethal to cells

  the hosts. In addition, modifications (e.g. glycosylation) and treatment (e.g. cleavage) of the protein products are important for the operation.

  of the protein. Different host cells have mechanisms

  specific and specific post-treatment

  translation and modification of proteins. Appropriate cell lines or host systems may be selected to ensure modification and treatment

  correct of the foreign protein expressed.

  In two particular forms of decri-

  In detail in the examples of the present invention, the chimeric LAV / HTLV III envelope coding sequences were ligated, both in their full form and in their portions, or LAV / HTLV gag coding sequences. III on the 7.5K promoter of vaccinia virus to form chimeric genes in various plasmids. We surrounded

  chimeric genes of these plasmids by additional sequences

  vaccinia virus homologs homologous to the vaccinia virus TK gene. The construction of the chimeric gene involved

  the use of nucleotides, both natural and syn-

  theories, coding control signals for the transcription of

  translation and translation of LAV / HTLV envelope sequences

  III or gag sequences of LAV / HTLV III. We then introduced

  These chimeric genes were generated in the vaccinia virus expression vectors by recombination in vivo between the homologous TK region present on the plasmid vector and the vaccinia virus genome, using these recombinant viruses containing the chimeric gene. as expression vectors for producing LAV / HTLV III-related proteins or producing gag-related proteins of LAV / HTLV

III.

  In other particular embodiments detailed in the examples of the present invention, the LAV / HTLV III envelope coding sequences or LAV / HTLV III gag coding sequences were ligated to the LAV / HTLV III gag coding sequence. polyhedrin of Autographa Californica nuclear polyhedrosis virus (AcNPV) for forming chimeric genes in various plasmids. The chimeric genes in these plasmids were surrounded by additional sequences of AcNPV. The construction of the chimeric genes involved the use of natural nucleotide coding control signals for 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, were used as expression vectors to produce LAV / HTLV III-related proteins or related proteins.

to gag of LAV / HTLV III.

  IDENTIFICATION OF RECOMBINANT EXPRESSION VECTORS

  CAPABLE OF REPLICATION AND EXPRESSION OF INSERTED GENE

  Three general approaches can be used to identify expression vectors containing inserted sequences of foreign genes: (a) DNA-DNA hybridization; (b) presence or absence of "marker" gene functions, and (c) expression

inserted sequences.

  Once a particular molecule of recombinant DNA has been identified and isolated, several methods can be used to propagate it, depending on whether such a recombinant

  is a self-replicating unit (a replicon)

  A self-replicating unit, for example plasmids,

  viruses, cells, etc., can multiply in the environ-

  cell and under appropriate conditions.

  required for growth. Recombinants missing a unit

  self-replication will have to be integrated into a com-

  carrying such a unit for propagation to take place. For example, certain plasmid expression vectors must, when introduced into a host cell,

  to be integrated into the cell chromosome Dour quarring the Dro-

  pagation and stable expression of the recombinant gene. Once a suitable host system and suitable growth conditions have been established, the propagation of the recombinant expression vectors can be propagated and prepared in quantity. In particular embodiments of the invention described in detail in the examples, chimeric genes containing the sequence encoding the coat or gag of LAV / HTLV III were inserted into the TK gene of the virus genome.

  vaccinia, thus transforming the virus into TK-, that is,

  that is, destroying the ability of the virus to make thymidine kinase. Such recombinants have been chosen according to

  their ability to grow in media containing 5-

  bromo-deoxy-uridine, which is a nucleoside analogous to the lethal one for TK cells but not for TK- cells. Recombinants have also been identified by DNA-DNA hybridization, using LAV / HTLV III envelope specific probes or LAV / HTLV gag specific probes.

  The recombinant TK virus is isolated by purification.

  plate and stockpiles were prepared from

  infected tissue culture cells.

  In other particular embodiments of the invention, described in detail in the examples, chimeric genes containing the envelope or gag coding sequence of LAV / HTLV III in the polyhedrin gene of the AcNPV genome have been inserted. , thereby transforming the virus into a non-occlusion phenotype. Such recombinants were visually selected based on their absence of occluded virus particles. Recombinants were further identified by Northern (Northern) coagulation analysis using probes

  specific LAV / HTLV III envelope or specific probes

  gag of LAV / HTLV III. Recombinant viruses were isolated by plaque purification and prepared

  reserve stocks from tissue culture cells

infected.

  IDENTIFICATION AND PURIFICATION OF THE PRODUCT OF THE EXPRESS GENE

  Once identified a recombinant that expresses the

  LAV / HTLV III gene, the gene product should be analyzed.

  This can be achieved through analyzes and assays based on

  physical, immunological or functional properties-

  the product. Immunoassay is particularly important when the ultimate goal is to use gene products or recombinant viruses that express such

  products in vaccine formulations and / or as

  tigenes in immunological assays for diagnostics.

  Various antisera are available to analyze the immunoreactivity of the product, which includes, but is not limited to, serum from patients with LAS (adenopathy syndrome) or AIDS and polyvalent antisera directed against LAV virus. / HTLV, viral coat protein or core proteins encoded by the gag gene. Immunogenic molecules include analogs

  produced in bacterial systems, or syngeneic peptides

  containing antigenic determinants of the envenom-

  LAV / HTLV III or LAV / HTLV III core antigens. The identification of the peptides and proteins described herein

  The invention is based on two requirements. First, the LAV / HTLV III-related protein is to be produced only in cells infected with recombinant virus. Secondly, the related LAV / HTLV III protein must be capable of immunological reaction with the serum of AIDS patients or with various antibodies directed against envelope or

  LAV / HTLV III core or their analogues and derivatives.

  -The protein must be immunoreactive, whether it results from the expression of the entire entire sequence of genes, a part of the gene sequence or two or more gene sequences that are linked by ligation to direct production fusion proteins. This reactivity can

  to be demonstrated by standard immunological techniques

  as a radioimmunoprecipitation, a competition

  radioimmunoassay or immunocoagles.

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

  Alternatively, once an immune protein has been identified,

  As a non-reactive related to LAV / HTLV III and produced by a recombinant, one can deduce the sequence of-amino acids of the immunoreactive protein according to 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 of the present invention, such peptides, whether produced by recombinant DNA techniques or by chemical synthesis methods, include, but are not limited to, all or one of the following: part of the amino acid sequences substantially as shown in Figure 2 or Figure 14, which includes altered sequences in which functionally equivalent amino acid residues replace residues within the sequence, creating silent variation. For example, one or more amino acid residues in the sequence may be replaced by another amino acid of similar polarity, which acts as a functional equivalent, giving a silent alteration. We can choose, among

  other members of the class to which the amino acid belongs

  holds, elements to replace an amino acid within

  the sequence. For example, non-polar amino acids (hydro-

  phobes) include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Neutral polar amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Amino acids (basic) to

  positive charge include arginine, lysine and histi-

  dine. Amino acids (acids) with negative charge include 4 n

  aspartic acid and glutamic acid.

  DETERMINATION OF THE IMMUNE POWER OF THE

RECOMBINANT PRODUCT

  The immune power 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 protein related to LAV / HTLV III is

  expressed by an infectious recombinant virus, it is possible to use

  ser the recombinant virus itself to immunize the test animals. The test animals may include mice, rabbits, chimpanzees, and possibly humans may also be used. Methods of introducing the immunogen may include intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,

  intranasal or any other classical route of immunization

  nization. The empirical response of the test subjects can be analyzed by three approaches: (a) the reactivity of

  the resulting serum immunity with respect to authentic viral antigens.

  LAV / HTLV III ticks, according to assays or tests carried out

  by known techniques, for example the immunoassay

  enzyme-linked sorption (ELISA), immunocoagulants,

  radioimmunoprecipitations, etc. (b) the ability of the

  serum to be neutralized "in vitro" the infectivity of LAV / HTLV III (M. RobertGuroff, 1985, Nature 316: 72-74) (for detection of anti-enveloped antibody), and (c) protection against

  ! infection - at LAVr, / HTLV III and / or attenuation of symptoms

  infectious diseases in immunized animals (D.P. Francis,

  1984, Lancet 2: 1276-1277; D.C. Gujdusek, 1985, Lancet 1: 55-56).

FORMULATION OF A VACCINE

  The purpose of this embodiment of the invention

  is to formulate a vaccine in which the immunogen is

  to an epitope of LAV / HTLV III or which contains a recombinant virus that expresses such an immunogen that protects against LAV / HTLV III virus infections for the prevention of lymphadenopathy (LAS) or AIDS. In addition, multivalent vaccine formulations that contain more than one LAV / HTLV III related immunogenic determinant can be prepared. Such vaccines include, but are not limited to, those containing

  epitopes related to the envelope of LAV / HTLV III

  isolated or in combination with other LAV / HTLV III epitopes, such as gag-related epitopes. Such multivalent vaccines, containing both coded epitopes

  envelope and coded gag, may be important for the pre-

  prevention of AIDS development. Non-symptomatic individuals seem to make anti-gag antibodies more often than sick patients, while both groups of patients make antibodies directed against envelope determinants (Science 1986, 233: 419). Studies have also shown that patients with AIDS produce, at early stages without symptoms, antibodies against envelope and core proteins (gag). As the disease progresses and symptoms appear, anti-gag antibodies are reduced, while anti-envelope antibodies remain (Science, 1986, 233282). Thus, inclusion of HIV-related epitopes, the production of which is described as a specific aspect of the invention, may be important for immunoprophylaxis or immunotherapy in

  the case of an illness related to LAV / HTLV III.

  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 adeno-

  disease (LAS) or AIDS and other diseases. Examples

  various formulations are presented hereinafter.

VIRUS VACCINE FORMULATIONS

  A live recombinant virus vaccine or an inactive recombinant virus vaccine can be formulated. The choice depends on the nature of the recombinant virus that is used to express LAV / HTLV III related epitopes. When the recombinant virus is infectious for the host to be immunized

  but does not cause disease, a live vaccine is preferred

  because a multiplication in the host leads to prolonged stimulation of a similar genus and amplitude

  what happens in natural subclinical infections

  and, therefore, this confers a strong long-term immunity. When introduced into a host animal, the virus

  infectious recombinant can express the relevant proteins

  to LAV / HTLV III from its chimeric gene and thereby stimulate an immunological response against LAV / HTLV III antigens. When such an immunological response provides protection against further attack by LAV / HTLV III, the live recombinant virus can serve itself

  preventive vaccine against infection with the AIDS virus.

  The production of such a recombinant virus for use in these formulations may involve systems both "in vitro" (e.g., tissue culture cells) and "in vivo" (e.g. 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 (eg, see Vaccine Viruses and Factors for Vaccine Antigens, published under

  direction of G.V. Quinnan at Elsevier, 1985, pp. 109-116).

  Multivalent live virus vaccines can be prepared from a single or a small number of viruses

  infectious recombinants that express several epitopes

  LAV / HTLV III / HTLV annuities. The vaccine may also include viruses that express epitopes of organisms causing other diseases, in addition to LAV / HTLV III epitopes. For example, a vaccinia virus (which can receive about 35 kp of foreign DNA base) can be genetically engineered to contain coding sequences.

  of LAV / HTLV III epitopes related to the envelope and gag.

  The virus may also encode other epitopes in addition to those for LAV / HTLV III. Such a recombinant virus itself may serve as an immunogen in a multivalent vaccine. Alternatively, a mixture of vaccinia virus or other viruses, each capable of directing the expression of a different coding gene, can be formulated in a multivalent vaccine.

  for different epitopes of LAV / HTLV III and / or other

  can cause disease.

  Whether or not the recombinant virus is infectious for the host to be immunized, an inactive vaccine formulation can be prepared. Inactivated vaccines are "dead" in that their infectivity has been destroyed, usually by formaldehyde treatment. In principle, the infectivity of the virus is

  destroyed without affecting the capsid or

  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. We can use

  be a mixture of inactivated viruses that express different

  epitopes to formulate "multivalent" vaccines. In

  In some cases, this may be preferable to live vaccine formulations, because of potential difficulties with

  to mutual interference of live virus administered - -

  together. In either case, it is advisable to formulate the inactive recombinant virus, or the mixture of such viruses,

  with a suitable adjuvant to amplify the immune response

  to their antigens. Suitable adjuvants

  take, without this list being exhaustive, gels

  minerals, for example aluminum hydroxide; subsi-

  surfactants such as lysolecithin, polyols "Pluronic"; polyanions; peptides; emulsions in oil and adjuvants potentially useful for humans, such as BCG (Bacille Calmette Guerin)

and corynebacterium parvum.

  Many methods can be used to introduce

  to obtain the vaccine formulations described above; they

  include, but are not limited to, intradermal

  intramuscular, intraperitoneal, intravenous, subcutaneous,

  cutaneous and intranasal. When using a live recombinant virus vaccine formulation, this vaccine can be introduced by the natural route of infection of the related wild-type virus that has been used to obtain the virus.

  recombinant in the formulation of the vaccine.

  FORMULATION OF SUBUNIT VACCINES

  Instead of virus-based vaccines, one can use

  the protein related to LAV / HTLV III itself as immune

  nogenous in subunit vaccine formulations that can be multivalent. As previously explained,

  subunit vaccines include only the immune

  involved, necessary to immunize a host. Thus, the related LAV / HTLV III protein (s) can or can be purified from recombinants that express LAV / HTLV III epitopes. Such recombinants include any of the virus-infected cultured cells, bacterial transformants, transformants and the like.

  yeast or insects infected with viruses that express

  LAV / HTLV III epitopes (see above for insertion of protein coding sequences, identification of recombinant expression vectors, and identification and purification of the expressed gene product). In another embodiment of the present invention, peptides or proteins related to LAV / HTLV III may be obtained by chemical synthesis. For this purpose, it is possible to deduce the amino acid sequence of such a peptide or protein from the nucleotide sequence of the chimeric gene which directs its expression (see, for example, what has been indicated below. about it about identification and

  purification of the expressed gene product).

  Whether the immunogens are purified from recombinants or obtained by chemical synthesis, the product

  final report may be adjusted to an appropriate and

  mated with any adjuvant suitable for vaccine,

  then packaged for use. Adjuvants

  Examples include, but are not limited to, inorganic gels, for example, aluminum hydroxide, surfactants such as lysolecithin, "Pluronic" polyols; polyanions; peptides; emulsions in oil and adjuvants potentially useful for humans, such as BCG (Bacillus Calmette Guerin) and corynebacterium parvum. The immunogen may also be incorporated into liposomes, or conjugated to polysaccharides and / or other polymers for use in formulation

of vaccine.

  When the peptide or protein related to LAV / HTLV

  III is a hapten, that is to say a molecule that is anti-

  Because the gene may react selectively with recognized antibodies, but is not antigenic because it can not elicit an immune response, the hapten may be covalently bound to an immunogenic carrier or molecule; for example, a big protein like

  Protein serum albumin will confer an immunological

  hapten gene coupled to it. We can formulate

  seems hapten-carrier for use as a vaccine.

  PASSIVE IMMUNITY AND ANTI-IDIOTYPIC ANTIBODIES

  Instead of active immunization with virus or subunit vaccines, it is possible to confer short-term protection on a host by administering preformed antibody to one or more LAV / HTLV epitopes. III. Thus, the vaccine formulations described above can be used to produce antibodies for use in passive immunotherapy. We prefer in medicine

  human immunoglobulin, because of the immunoglobulin

* heterologous line may cause immune response

  to its foreign immunogenic components. Such immunization

  passive intervention could be used in case of emergency for

  Immediate exposure of non-immune persons exposed to special risks, for example those exposed to contact with AIDS patients, for example, in hospitals and other health facilities. Alternatively, these antibodies can be used to produce antiidiotypic antibody, which in turn can be used as an antigen to stimulate an immunological response against

epitopes of LAV / HTLV III.

IMMUNOLOGICAL ASSAYS

  The peptides and proteins of the

  the present invention, related to LAV / HTLV III, as anti-

Z587720

  genes in assays and immunoassays for the detection of

  antibodies against LAV / HTLV III in a variety of patients' body tissues and fluids, as well as blood from blood banks and hospitals. For this purpose, the antigens of the present invention can be used in any immunoassay system known in the art.

  includes, but is not limited to, radioimmunoassays,

  the so-called "ELISA" assays, "sandwich" assays, precipitation reactions, reactions

  precipitation by gel diffusion, the immunoassay

  diffusion, agglutination assays, complement fixation assays, immunoradiometric assays,

  immunofluorescence assays, immunoassays,

  protein A and immunoelectrophoresis assays,

to name a few.

  The immunoassays and assays of the present invention can be used to perform blood tests of blood banks and patients for

  to detect possible exposure to LAV / HTLV III and to

  ensure patients who are treated because of LAS or

  AIDS. In the assay or immunoassay,

  logic according to the invention, any peptide or any

  which protein is related to a LAV / HTLV III antigen.

  Such antigens include, but are not limited to, peptides and proteins related to env gene products, gag, pol and the four additional genes so far designated: sor, tat, 3'-orf and art (or very). . In a particular embodiment of the present invention, a set of antigens can be used in an immunoassay assay to screen patients for antibody profile profiling against said different LAV / HTLV epitopes. III. In another embodiment of the invention, patients who have been vaccinated with a vaccine formulation can be monitored.

  of the present invention for detecting

  quente to LAV / HTLV III. In such cases, the antigen used in the immunoassay is preferably a peptide or protein of the invention that was not an immunogen of the vaccine formulation used to vaccinate the patient, as such vaccinated persons will be seropositive. for the particular epitope used in the vaccine formulation and, therefore, will give a positive test result, whether or not there has been exposure to the virus. For example, if

  a patient has been vaccinated with an epitope of the present invention.

  LAV / HTLV III-related envelope, this patient may be screened for LAV / HTLV III infection tests using any other protein unrelated to LAV / HTLV III. envelope of LAV / HTLV III, to determine the presence in the patient of specific antibodies for LAV / HTLV III. Thus, a patient vaccinated with an envelope-related epitope may be screened for antibodies specific for LAV / HTLV III nuclei antigens, or other LAV / HTLV III antigens, including, without being limiting

  the products of the sor and 3'-orf genes and the like.

  In another embodiment, it is possible to use

  Serum peptides and proteins of the invention, related to LAV / HTLV III, in competitive immunological assays, to detect and quantify the presence of proteins encoded by LAV / HTLV III-HTLV III, in blood, serum, etc. , of a patient.

  EXAMPLE: RECOMBINANTS OF VACCINE ENVELOPE

  In the following examples, various plasmid vectors containing chimeric genes including downstream LAV envelope coding sequences with respect to the transcription control sequences of the present invention were constructed.

vaccinia virus.

  These chimeric genes containing the vaccinia promoter and the LAV / HTLV III envelope coding sequence were inserted into the genome of the vaccinia virus by in vivo recombination. Such recombinant viruses have been identified

  purified and stockpiles of the virus have been prepared

  res from infected tissue culture cells.

  Immunoreactive proteins, related to the LAV / HTLV III envelope, have been shown to be produced by these viruses.

  recombinants of vaccinia in vitro. These recommendations were

  test animals in order to determine their ability to trigger immunological neutralization or protection responses and to study their use.

  as AIDS vaccines. A description

  detail of each step of this embodiment of

  the invention is presented below.

  Here are some details about the procedures

General.

CELLS AND VIRUSES

  From R. Condit (Associate Professor, Department of

  Biochemistry, New York State University, Buffalo, New York, USA) African green monkey kidney cells (strain BSC-40, continuous line of green monkey cells from Africa from BSC-1 cells, n ATCC CCL26), and propagated in Dulbecco's modified Eagle's medium (MEMD, Gibco, Grand Island, New York) supplemented with 10% fetal serum. of cattle and 100 units of penicillin and 100 units of streptomycin

  per milliliter. It was obtained from Mr. Botchan (Professaur, Depar-

  Molecular Biology, University of California, Berkley, Calif., USA) of human TK cells (JS Rhim et al., 1975, Int. J. Cancer 15: 23-29) and was challenged therein. propagation in the above medium, with the addition of 5-bromo-deoxyuridine (BUdR)

at 25 μg / ml.

  We obtained American Type Culture Collec-

  (ATCC n CCL 171) a human diploid cell (MRC-5) which has been propagated in the same medium as that

used for BSC-40.

  Raccid viruses (strain WR, n ATCC VR119) were obtained from R. Condit and cultured in BSC-40 cells in MEMD + 5% gamma-free globulin serum + 100 units / ml. penicillin and 100 units / ml of streptomycin. TK recombinants were selected from TK cells in the same medium containing 25 μg / ml BUdR and plaque purified on the same cell line in MEMD containing 1% Noble agar (DIFCO, Detroit, Michigan). , United States of America), 5% gamma globulin-free calf serum, 100 units / ml penicillin and 100 units / ml streptomycin and 25, g / ml BUDR. virus in phosphate buffered saline containing 8 g of NaCl, 0.2 g of KCl, 1.5 g of NaH2PO4, 0.2 g of K2HPO4 per liter, with addition of 1 mM MgCl2 and 0.01 per liter % of

serum albumin of cattle.

  The strain of vaccinia virus from the New York City Board of Health

  the city of New York) has been purified from a

  commercial application of smallpox vaccines ("Dryvax") (R), (batch 321-501 g) sold by Viet Laboratories (Marietta, PA, United States of America). The smallpox vaccine was diluted with phosphate buffered saline with various adjuvants (see below) and subjected to plaque purification three times successively using BSC-40 cells. A stockpile, hereinafter referred to as vNY, was prepared on BSC-40 cells from such a plaque-purified isolate and was used to construct

  recombinant viruses. It was ensured in MRT-cells

  5000, the spread of recombinant virus stocks from

from v-NY.

  Refer to the main patent for the descrip-

  more detailed information on the preparation, restriction and modification of

  DNA, vaccinia virus-containing plasmid vector constructs, promoter, ligated to LAV / HTLV III envelope gene coding sequences and plasmid vector constructs containing virus. vaccinia promoter linked by ligation to the sequence

  3 'or 5' coding of the LAV / HTLV III envelope gene.

  CONSTRUCTION OF PLASMID VECTORS CONTAINING VIRUSES

  PROMOTER VACCINE CONNECTED BY LIGATION TO ALL

  ENCODING SEQUENCES OF LAV / HTLV III ENVELOPE GENE

  Two micrograms of plasmid pv-envl DNA were digested until completion with StuI restriction enzyme PvuI and XhoI. The resulting fragments were resolved on a 1% low melting temperature agar gel. The

  cleavage of Pv-envl using StuI and PvuI gives two fragments

  4 pKb, one containing the 5 'portion of LAV / HTLV III and the other containing pGS20; XhoI cleaves the 4kbp fragment containing pGS 20 into smaller fragments which, thus, allow identification of the StuI / PvuI 4kbp fragment containing the virus transcriptional control element

  vaccinia and the 5 'part of the enzymic coding sequences.

  loppe of LAV / HTLV III. This fragment was isolated, purified and ligated to a 6.5 kbp fragment generated by restriction enzyme digests StuI and PvuI of pv-env2. The ligation mixture was used to transform E. coli strain MC1000. Transformants that can withstand ampicillin have been chosen. Plasmid DNA from individual transformants was subjected to assays for the determination of StuI and PvuI restriction site regeneration and the presence of 4 kilobase (4 pKb) and 6.5 pKb related fragments. The desired plasmid, pv-env5, depicted in FIG. 4, contains the entire LAV envelope gene corresponding to nucleotides # 5766 to # 8349 (as shown in FIG. 2), connected by ligation downstream of the control elements of

  transcription of the vaccinia virus.

  CONSTRUCTION OF PLASMID VECTORS CONTAINING PROMOTER

  OF VACCINE VIRUS RELATED BY LIGATION TO ENVELOPE GENE

  OF LAV / HTLV III WITHOUT THE TRANSMEMBRANE SEQUENCE (ANCHORAGE)

  Digestion of 10 μg of plasmid DNA was

  Pv-env5 to completion, by HIND-3, and the resulting fragment resolved on a low melting 1% agar gel. The fragment with 3 kilograms was purified

  the 7.5 K vaccinia promoter and the promoter

  5 'LAV / HTLV III envelope coding sequences,

  then they were treated with Klenow's enzyme to supplement

  ter the cohesive ends. This fragment was then ligated to a multi-site cloning vector, P26, subjected to

  prior to digestion with EcoRI, then treated with Klenow enzyme and with phosphatase

  alkaline of calf intestines. A ligation of the site of 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 trans-

  form E. coli MC1000, amplify it and purify it. It was then digested with BamHI and HpAI, and the resulting fragments were resolved on a 1% agar gel. The 2.1 kilobase base fragment containing the LAV / HTLV envelope coding sequence was isolated, and then ligated onto pGS 20 plasmid submitted.

  before digestion with BamHI and SmAI. The plasmid Pv-

  Resultant env 7 contains the 7.5 K vaccinia promoter ligated to a LAV / HTLV III specific sequence from the 96 base pairs upstream of the envelope priming codon from the HIND 3 site. at nucleotide number 6798 (Figure 5). This plasmid therefore does not include the presumed sequence of anchoring the coat envelope gene.

LAV / HTLV III.

  CONSTRUCTION AND CHARACTERIZATION OF VACCINE VIRUS

  RECOMBINANT CONTAINING THE CHIMERIC GENE OF ENVELOPE LAV /

HTLV III

  Two vaccinia virus strains were used for the construction of the recombinant viruses: WR, the research control strain, and v-NY, a derivative of the strain from the New York City Board of Health.

  Public Health of the City of New York).

  The insertion of the chimeric gene sequences corresponds to

  LAV / HTLV III enveloping in the genome of vaccinia virus is obtained by "in vivo" recombination, made possible by the fact that the chimeric genes are surrounded in the plasmids pv-env2, pv-env5 and pv -env7 by

  sequences of the vaccinia virus encoding the thyroid gene

  midine kinase (TK). Introduction of these plasmids into vaccinia virus infected cells 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 a double recombination

  naison in the lateral entourage sequences. Such recombinants will include the chimeric gene inserted into the gene

  TK of vaccinia and, therefore, their phenotype will match & TK.

  These TK recombinants can be chosen to grow in a medium to which BUdR has been added, which is lethal to

  TK + cells but not for TK- cells. The principle

  The general principle of this procedure has been described (Mr Mackett,

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

  CONSTRUCTION OF THE RECOMBINANT VACCINE VIRUS CONTAINING THE

  CHIMERE GENE CORRESPONDING TO LAV / HTLV III ENVELOPE

  A box 100 mm in diameter was infected

  carrying 80% confluent African green monkey kidney cells (strain BSC-40) with vaccinia virus (strain WR) at a multiplicity of infections of 0.05. After 2 to 4

  hours of incubation at 37 C, the cells were covered

  by coprecificities, using calcium phosphate

  cium, pv-env2 or pv-env5 plasmid DNA. We prepared

  precipitates by adding dropwise 0.5 ml of a solution.

  2XADN-CaC12 to 0.5 ml of 2XHeBS solution (2XADNCaC12 solution contains 20 μg of plasmid DNA in 0.5 ml of 0.25M CaC12; 2XHeBS contains, per ml, 16 mg of NaCl, 0.74 mg KCl, 0.25 mg Na2HPO4 2H20, 2 mg dextrose, and 10 mg HEPES, pH 7.08). The coprecipitates obtained with calcium phosphate DNA were allowed to form at room temperature for 30 minutes. Four hours after having covered with precipitate, the cells were washed once with 1XHeBs, incubated at 37 ° C. for 3 minutes in the presence of 2 ml of 15% glycerol in 1XHeBs,

  then washed again with 10 ml of growth medium.

  (MEMD + 10% fetal calf serum + 100 units / ml penicillin and 100 units / ml streptomycin). Two days later, the infected cells were harvested and

  collected by centrifugation (40C, 10 minutes & 2010 x g).

  25877zi0 Virus stocks were prepared by putting these cells to

  again in suspension in 1 ml of a saline buffer solution

  bovine serum albumin, followed by two cycles of freezing and thawing and three

ultrasound.

  Recombinants were selected by placing 0.1 ml of the 10-fold dilution of the virus stocks from the above operations on circular plates in 60 mm boxes of TK confluent human cells, topped with 5 ml / dish of 1% Nobel Agar (Difco, Detroit, Michigan) with 5% calf serum, 25 μg / ml BUdR in MEMD. Two days later, the cells were stained by placing 2 ml / dish 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 and supplemented with bovine serum albumin (PBSAM), and used aliquots (0.25 mi) of the virus suspensions to infect TK confluent cells placed in 16 mm diameter depressions in a selective medium (MEMD +

  % fetal calf serum + 100 units / ml streptomycin

  cine + 100 units / ml penicillin + 25 μg / ml BUdR). Infected cells were collected by centrifugation and resuspended in 100 μl phosphate buffered saline (PBS) containing 0.5 mg / ml trypsin and 0.2 mg / ml EDTA (ethylene acid).

  tetraacetic diamine). The cells were lysed by incubation

  at 37 C for 30 minutes, followed by 3 cycles

  each 20 seconds of sonication treatment. We have

  on nitrocellulose filters, cell lysates

  the, using a manifold or filtration manifold

  several troughs (Schleicher and Schuell, Arlington, MA).

  DNA-DNA hybridization was determined as described (M. Mackett, G. L. Smith and B. Moss, 1982, Proc Natl Acad.

  Sci. 79, 7415-7419), the presence of the specific DNA sequences

  of the LAV / HTLV III envelope in these samples.

  Hybridization probe used plasmid DNA

  pRS-3 labeled with 32p, prepared by appropriate translation.

  Recombinants which gave positive hybridization for this probe, on TK cells under selective conditions (medium containing BUdR) and once on BSC-40 cells under non-selective conditions were further purified twice on plate. After final confirmation by DNA-DNA hybridization, virus stocks were prepared from the three-fold purified plaques on cells

  BSC-40 and used for subsequent characterization.

  quente. A schematic representation of the construction of recombinant viruses is shown in Figure 6, in which the gene sequence corresponding to the LAV / HTLV III envelope (env) located downstream of the 7.5 K vaccinia promoter (p- ) is surrounded within the vaccinia genome by TK DNA sequences. Virus stocks from recombination between the vaccinia virus genome

  and the plasmid pv-env, were called v-env5, and they were

  hold the entire LAV / HTLV III envelope gene.

  In the particular form of implementation described in

  examples here, v-env5 contains the entire enve-

  as well as 96 base pairs of non-translated sequences close to 5 'and 223 base pairs of non-translated sequences close to 3'. Virus stocks from

  of pv-env2 have been called v-env2 and they contain the most

  part of the LAV / HTLV III envelope gene (i.e., the KpnI fragment), but they do not include the portion of the sequence encoding the first 42 amino acids of the LAV envelope protein. / HTLV III. Since the supposed signal sequence of the LAV / HTLV III envelope protein is located within the first 49 amino acids of the protein, v-env2 should produce a protein lacking the signal sequence; therefore, the LAV / HTLV III related protein produced by this virus should be expected

  recombinant is not transported to the membrane. At the

  milk, v-env5, which contains the entire envelope sequence of LAV / HTLV III must produce a protein that

  is transported to the membrane. Stocks of viruses from

  pv-env7 are referred to as v-ehv7 and contain the complete nitrogen termination of the gene corresponding to the LAV / HTLV III envelope, but do not include the sequence

  downstream of the HIND III site. Since the presumed sequence trans-

  membrane (anchorage) is missing in this construct, one would expect that the LAV / HTLV III related protein, produced by this recombinant, would 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 vaccine

with subunits.

  FORMATION OF RESTRICTION FRAGMENTS OF RECOMBINANTS

VACCINE ENVELOPE OF LAV / HTLV III

  As described previously (Esposito et al., 1981, J. Virol, Methods 2.2, 1975-180) the strains of the starting vaccinia virus WR and v-NY as well as their v-env5 and v-env5NY, respectively. We submitted them

  digestion with the restriction enzyme HIND III and the fragments

  The resulting cultures were resolved on a 0.7% agar gel.

  the DNA from the starting strain WR presented the model

  typical restriction fragment (FIG. 2A) as previously

  described (De Filippesy 1982, J. Virol 43: 136-149). The restriction fragment pattern for v-NY was similar, but distinct from that obtained with WR. The most obvious differences lie in the length of fragments b and c obtained with HIND III (HIND IIIb and c fragments), which are terminal fragments of the vaccinia virus genome. The recombinants v-env5 and v-env5NY do not contain HIND III J fragments of their starting or parental strains. Instead, two new fragments were observed,

  having a size of 5.4 Kbp (kilopiles of bases) and 3.2 Kbp.

  This observation is consistent with the result of the double recombination that inserted the sequence encoding the LAV / HTLV III envelope into the vaccinia TK gene, which is located on the HIND III fragment J. Two fragments were generated by insertion. because of the presence of a HIND III site in the LAV / HTLV III sequence. This result also confirms the orientation of the inserted LAV / HTLV III fragment with respect to the vaccinia virus TK gene. ANALYSIS BY COAGULATION TYPE "SOUTHERN" (South) DE

  RECOMBINANTS VACCINE-ENVELOPE LAV / HTLV III

  The identity of two new HIND III fragments in the restriction digests of recombinant viral genomes was confirmed by Southern coagulation analysis. DNA fragments, resolved on a gel of

  as shown in Figure 7A, have been trans-

  on nitrocellulose filters, and hybridized on a probe obtained by specific translation of 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 represented on the

figure 6.

  EXPRESSION OF PROTEINS RELATED TO THE ENVELOPE OF LAV /

  HTLV III IN INFECTED TISSUE CULTURE CELLS

  BY RECOMBINANT VACCINE VIRUSES

  Recombinant vaccinia viruses, harboring LAV / HTLV III envelope chimeric genes, have been shown to be capable of expressing LAV / HTLV III envelope-related proteins upon cell infection in a culture. tissue. These proteins have also been found to be immunoreactive with patient-derived serum.

with AIDS.

  Refer to the main patent for the descrip-

  detailed description of the identification, using techniques

  immunocoagulation or immunoprecipitation of

  related to the envelope of LAV / HTLV III expressed in

  cells infected with recombinant vaccinia virus.

  It is noted that the results obtained are shown in FIGS. 8A and 8B of the present disclosure, corresponding to FIGS. 6A and 6B of the main patent (immunocoaqulation) and

Figure 9A (immunoprecipitation).

J

  3H-GLUCOSAMINE MARKING OF PROTEINS RELATED TO

  LAV / HTLV III ENVELOPE PRODUCED BY VIRUSES

  VACCINE RECOMBINANTS CORRESPONDING TO LAV / HTLV III

  The radioimmunoprecipitation assay described in the main patent gave results shown in Fig. 9B (corresponding to Fig. 7B of the main patent),

  however, the results of the immunoprecipita-

  with hunting drives, proteins related to

  the envelope of LAV / HTLV III produced by recombinant viruses

  Vaccines related to LAV / HTLV III, described in detail in the main patent, appear in Figure 9C (corresponding

  in Figure 7C of the main patent).

  PRESENCE OF A PROTEIN RELATIVE TO THE ENVELOPE OF LAV /

  HTLV III IN THE GROWTH ENVIRONMENT OF INFECTED CELLS

  BY RECOMBINANT VACCINE VIRUSES-LAV / HTLV III

  The radioimmunoprecipitation assay described above

  afterwards showed that the immunoreactive proteins produced by the recombinant vaccinia virus of the invention can

  expressed and treated in a manner similar to glycoprotein

  authentic LAV / HTLV III envelope teat.

  Confluent monolayers of HeLa cells were "infected" with non-infectious virus (see Figure 9D, lane 1) or separately infected with wild-type vaccinia virus (lane 2), with recombinant v-env5

  (lane 3) or recombinant v-env2 (lane 4), all at one

  tiplicity or infection rate of 20 training units

  plate per cell. The cells were labeled with 35 S-methio

  nine (more than 800 Ci / mmol, Amersham) at 100> Ci / ml 10-12 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 using it for an immunization

  noprecipitation with pooled serum from persons with a positive result for LAV / HTLV III. Proteins from infected cells immunoprecipitated with the same serum are presented in the pelletized portion and proteins from the growth medium or culture.

  are shown in the section marked "Supe" (upper middle

laughing supernatant).

  As expected for gpl10 of LAV / HTLV III, the 120 kd protein obtained using the recombinant was also detected in the infected medium (FIG. 9D, FIG.

  track 3). These results showed that v-env5 virus recom-

  binant was able to express the gene corresponding to the

  veloppe of LAV and produce immunoreactive proteins that have been treated similarly to authentic LAV / HTLV III envelope glycoproteins. As would be expected for proteins lacking signal peptides LAV / HTLV III envelope-related polypeptides were not detected in the growth medium of v-env2 infected cells (FIG.

9D, lane 4).

  In addition, cells infected with v-env7 expressed a truncated protein of 130 Kd. This protein does not include the 217 amino acid end-carbons containing the LAV / HTLV III coat protein anchor sequences. This truncated protein (gp130) was secreted into the growth medium much more efficiently than gpl10 or its gp150 precursor (Figure 9E). At the same time, gp130 is not efficiently transformed into gp110, although the site of

  cleavage is still present on the truncated molecule.

  IMMUNE POWER OF THE RECOMBINANT VIRUS

CORRESPONDING TO THE ENVELOPE OF LAV

  Recombinant viruses, carrying the chimeric gene corresponding to the LAV / HTLV III envelope, have been shown to be capable of eliciting an antibody response against LAV / HTLV III in two mouse strains and a mouse species.

subhuman primate.

  IMMUNOGENICITY OF VACCINE RECOMBINANTS CARRYING THE GENE

  LAV / HTLV III ENVELOPE IN MOUSE

  Immunogenicity of proteins was examined

  expressed by v-env5 and v-env2 of vaccinia virus recom-

  binants. The experiments are described in detail in the main patent, which also indicates the results of the

  ELISA tests on 6 week serum samples.

  IMMUNOGENIC CHARACTER OF RECOMBINANT V-ENV5

  OF VACCINE-LAV / HTLV III VIRUS IN SUB-HUMAN PRIMATES

  Immunogenicity of vaccinia-V was studied

  recombinant env5 of LAV / HTLV III in two species of subhuman primates: Macaca Fasciculariès and chimpanzees. The results described below show that v-env5 is able to elicit in both species a humoral immunity and

cell-mediated.

  Humoral response in macaques monkeys immunized with v-env5 Nine long-tailed macaques (Macaca Fascicularis), ranging from youth to adulthood, were used to study the immunogenic character of recombinant vaccinia v-env5. All animals were subjected to preliminary screening tests for the absence of antibodies against vaccinia virus and LAV / HTLV III and the absence of monkey T-lymphotropic virus or monkey or AIDS virus. antibodies against these viruses. To four of the monkeys, 2 x 10 8 v-env plate formation units were inoculated and to 4 monkeys 2 x 10 plaque forming units of the same virus were inoculated. To one animal, 2 x 107 units of plaque formatin a vaccinia virus were inoculated

  recombinant control (v-HSgD1 of recombinant vaccinia virus

  herpes simplex gD1). All animals were inoculated by scarification of the skin. Serum and heparinized blood samples were collected prior to inoculation and after 4, 6 and 8 weeks after inoculation. Ten weeks after the first inoculation, all

  animals a second inoculation of 2 x 108 units of

  plate of the same virus. All.-the animals presented

  self-healing of skin lesions, typical of infections

  by the vaccinia virus, and physiological indications

  normal logic throughout the course of the experiment.

  The humoral response was analyzed by ELISA and by assays

  Western-type coagulation assay.

  For an "ELISA" analysis, the samples were diluted

  Serum 50 times in PBS containing 3% powder

  non-fat milk and 0.2% NP-40 (polyoxyethylene (9) p-tert-

octylphenol), and reacted with LAV / HTLV III virion proteins which had been immobilized on microtiter plate wells. The procedure for ELISA was identical to that described above, except that the second antibody used was anti-human antibody.

  of goat conjugated with horseradish peroxidase.

  The results obtained, presented in FIG.

  show that if only two animals have presented a serotype

  conversion after primary inoculation, all animals have

  presented with seroconversion after the second immunization.

  To determine which antibodies were produced in the immunized animals, serum samples collected at 4 weeks after the second immunization were analyzed by Western coagulation. We used

  two protocols for optimal detection of both glyco-

  envelope proteins, gp41 and gpl10. For optimal detection of gp41, serum was diluted 50 fold in PPS + 3%

  unpasteurized milk and 0.2% NP-40, and

  Leave for 1 hour at room temperature with purified LAV / HTLV III virion protein immobilized on a nitrocellulose filter. The filters were washed with PBS (physiological saline solution supplemented with phosphate buffer) + 0.2% NP-40, and reacted with

  goat anti-human immunoglobulin antibody

  treated 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 once 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 reacted with

  goat anti-human immunoglobulin conjugate

  alkaline phosphatase (Zymed, CA) b diluted

  from 1 to 2000 in PBS buffer. In each operating mode

  the final wash after the second reaction with the anti-

  body was made in 0.1M TRIS (pH 9.5) with 0.1M NaCl and 5mM MgCl 2. The antigen-bound antibody was detected on the filter by reacting the filter with a solution containing 0.1M Tris (pH 9.5), 0.1M NaCl, 5mM MgCl 2, 0.33g / ml of bromo-chloroindolyl phosphate and 0.17 mg / ml of nitrotetrazolium blue. After having done

  react the filters with the chromogens, we rinsed the filters

  very in water, air dried and photographed.

  As shown in Figure 12B, all animals that received two inoculations showed a strong reaction

  of antibodies to gp41. In addition, four of these

  evils have also produced antibodies that have strongly

  reacted with gpl10. As a whole, the results represent

  12A show that recombinant v-env5 is able to elicit, in macaque monkeys, the production

  antibodies specific for the LAV / HTLV III envelope.

  IMMUNOLOGICAL RESPONSE, WITH CELLULAR MEDIATION, IN

IMMUNIZED MACAQUES 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 vaccinia virus control expressing the glycoprotein

  herpes simplex virus (D-HSVgD1) dye, by centrifugation

  on "Ficoll Hypaque" device. Peripheral blood lymphocytes from macaque 81, which was vaccinated once only with v-env5, and from unimmunized macaques were also isolated. 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 place 1 x 105 peripheral blood lymphocytes

  in a final volume of 0.1 ml of the medium in plate cavities with 96 round-bottomed cavities. In each

  0.1 ml of medium containing v-env5 was

  by light (ultraviolet) (1 x 106 units of

  plate / ml before ultraviolet inactivation) or

  Unbroken LAV / HTLV III (1 mg / ml, approximately 1 x 105

  average tissue culture obtained), which was purified by two cycles of sucrose gradient centrifugation to separate the supernatant fluids from LAV / HTLV III infected CEM cells, which is a negative T cell leukemia cell line. HLADR type. Six days after the stimulation, each trough of peripheral blood lymphocytes was labeled with 1 μCi of 3H-TDR (3H-thydimine, New England Nuclear, Boston, MA, USA) for 6 hours, harvested. cells, and counts per minute (cpm) corresponding to incorporated 3H-TDR were determined as the average value obtained for four troughs. The stimulation index was calculated by dividing the number of strokes per minute corresponding to 3H-TDR incorporated into

  stimulated cells, by the number of strokes per minute corresponding to

  spawning when incorporated into unstimulated cells.

TABLE I

  Response, by LAV / HTLV III-induced proliferation, of peripheral blood lymphocytes taken from macaques, after immunization with recombinant vaccinia virus expressing the envelope glycoproteins

LAV / HTLV III

  Inr unisation v-env5 v-env5 v-env5 v-env5 v-env5 v-env5 v-env5 v-env5 venv5 V-HSVgD1 none none, cpm * Virus used for

LAV / HTLV III

cprn * IS **

7465 4.7

9075 4.0

4732 3.0

8645 14.9

5987 2.5

13922 12.9

3985 4.1

8580 3.3

553 1.0-

1822 1.0-

1072 1.0-

  stimulation of lymphocytes v-env 5 cpmf IS ** 38.1 12.8 13.6 14.1 14.8 23.0 42.0 18.1 91.9 1.0- 2.0 strokes per minute corresponding at 3H-TdR Incorporated stimulation index ro Ln o -j. N1 'Macaque no * cpm:

** IS:

  As shown in Table I, all immunized macaques, including # 81, which received a single inoculation of v-env5, produced lymphocytes that proliferated in specific response to LAV stimulation. / HTLV III in vitro. In contrast, lymphocytes from animal 73, which received recombinant herpes simplex recombinant vaccinia-v-HSVgD1, responded only to stimulation with

  vaccinia virus, but not to LAV / HTLV III.

  Non-immunized control animals did not produce lymphocytes

  cysts responding to either antigen. In addition, the response for lymphocytes from v-env-5 immunized macaques was similar in magnitude to that obtained for macaque 73, which was vaccinated with vHSVgD1. These results indicate that the expression of the LAV / HTLV III env gene by the recombinant virus did not suppress the cell-mediated immunological responses in macaque monkeys.

T, in vitro or in vivo.

  To determine the subgroup of lymphocytes

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

TABLE II

  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 a recombinant vaccinia virus expressing enteric glycoproteins.

  LAV / HTLV III lymphocyte * stimulated with v-env5% total cells

IL-2R +

  27.7 v-env 5% of IL-2R + cells coexpressing CD4 48.6, 62.4 CD9 CD20 (Bp35) 52.2, 0 2.0 NT

LAV / HITLV III

14.7

LAV / HTLV III

  , 0 12.0 no virus 58.0 0.3 0.3- 59.0 38.0 0.3- 1.9 1.5 0.3- * lymphocytes: peripheral blood lymphocytes * NT: no 'On 0o' test J Macaque n G \ ln The results of the table were obtained as follows.

  II. Simulated peripheral blood lymphocytes were stained

  at the same time with phycoerythrin-conjugated monoclonal antibody 2A3 (Becton-Dickinson, Inc., Mountain View, CA, United States of America), antibody to the receptor.

  interleukin-2 (IL-2R), and with monoclonal antibodies

  Fluorescein-conjugated IF5 clones (Genetic Systems Corp., Seattle, WA, USA) for CD20, G10-1 (Genetic Systems Corp., Seattle, WA) for CD8 or T4 (Ortho a

  Diagnostics, Raritan, N.J.) for CD4. We used the

  body at saturation concentrations, determined in advance by assays on lymphocytes analyzed by flow microfluorometry with a FACS classifier

  IV modified (Becton-Dickinson, Mountain View, CA). We have

  killed a quantitative two-color assay as previously indicated (Ledbetter, J.A. et al., 1984, Perspectives in Immunogenetics and Histocompatibility, 6, 119-129). The dispersion gates were adjusted forward and at right angles to include lymphoblasts and a large number of small lymphocytes. The values indicated are those concerning 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

  by antigen, produce lymphokines comprising

  IL-2, which can promote differentiation and / or

  fission of T and B cells and can activate natural lytic cells or globules able to lyse cells

  infected with various viruses, including the AIDS virus.

  It was therefore questioned whether the globules or T cells from vaccinated 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 injection of v-env5 or v-HSVgD1 immunization constructed with the WR strain of vaccinia virus. For experiment 2, peripheral blood lymphocytes were isolated from macaques 4

  weeks after a first intradermal injection of

  2 x 105 v-env5, v-HSVgD1 or v-env5 recombinant virus constructs constructed with the vaccine strain of the New York City Board of Health. New York City) (v-env5NY). Peripheral blood lymphocytes were suspended in RPMI 1640 medium supplemented with 10% normal heat-inactivated human serum and 2 x 10 5 peripheral blood lymphocytes were placed in 96-well hollow fund wells. round. We then

  added in hollow v-env5 inactivated by ultraviolet light

  violet (1 x 106 units of plaque formation / ml before inactivation by ultraviolet light) or purified LAV / HTLV III (1 μg / ml). Two days after the stimulation, the supernatant liquids were collected on hollows used in pairs, and assays were performed to determine the ability to maintain IL-2-dependent CTLL-2 cell proliferation (cells supplied by Dr. S. Gillis, Immunex Corp. Seattle, WA, USA), which was washed for 24 hours, prior to testing, to rid them of IL-2. During the last six hours of incubation with the supernatant, the cells were labeled with 3HTdR, and the amount of 3H-TdR incorporated into the cells was determined. The units of IL-2 activity present in the supernatant liquid, as described, were calculated from reference curves obtained by testing the effect of recombinant human IL-2 (provided by Dr. Gillis) on the proliferation of CTLL-2 cells. The

  The results obtained are presented in Table III.

TABLE III

  IL-2 production by peripheral blood lymphocytes from vaccinated macaques after stimulation with LAV / HTLV III or recombinant vaccinia viruses expressing AIDS virus envelope glycoprotein Experiment 1 IL-2 (units / ml ) Macaque no Immunization Vaccine-stimulated lymphocyte * supernatant fluids VLV / HTLV III v-env5 v-env5 v-env5 v-env5 v-HSVgD1 nil ooo O o 28.0 16.0 9.0 o 96.0 124.0 52.0 108.0 o Experiment 2: v-env5 v-env5NY v-env5NY v-env5NY v-HSVgDl nil nil 14.4 16.8 7.1 2.4 ooo, 8 33.3 26, 7 27.6 27.6 oo 27 :,

26 - ..,

oe CD ru o -4 rO> c>

  The results of experiment 1 of Table III

  that supernatant cells from v-env5 or LAV / HTLV III stimulated peripheral blood lymphocytes obtained on v-env5-immunized macaques contain IL-2, as shown by their ability to induce cell proliferation. CTLL-2, which is an IL-2 dependent cell line. Similarly, as shown in Experiment 2 of Table IV, IL-2 was detected in supernatant of LAV / HTLV III or v-env5 stimulated peripheral blood lymphocytes from Macaque 03, which was immunized with once with v-env5, and from all three macaques that were once immunized with the "vaccine" strain of the same recombinant (v-env5NY,

  see above the presentation on the construction and characteris-

  of the recombinant vaccinia virus containing the gene

  chimeric envelope of LAV / HTLV III). On the contrary, the

  peripheral blood phocytes from macaques 73 and 59, which were immunized with recombinant vaccinia-HSV gD-1 viruses, produced IL-2 after

  v-env5 stimulation only, and not after stimulation.

  by LAV / HTLV III. The unimmunized macaques 26 and 27 did not produce detectable IL-2 after stimulation with LAV / HTLV III or recombinant vaccinia virus. Since T cells or cells exhibiting helper / inducer 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 with recombinant viruses. In

  more of their probable role in the differentiation of

  or B cells to produce antibodies against

  LAV / HTLV III envelope antigens, cells or lymphocytes

  T-cells producing IL-2 may be involved in the differentiation and / or expansion of effector cells,

  like cytotoxic T lymphocytes or lytic cells

  natural diseases that can kill cells infected with

viruses.

  HUMORAL RESPONSES IN IMMUNIZED CHIMPANZES

V-ENV5NY

  To two juvenile chimpanzees, 5x10 8 plaque formation units of v-env5NY were intradermally inoculated, the "vaccine" strain of venv5. To an animal, we

  inoculated intradermally with the same title of a recom-

  vaccinia-herpes simplex gD bovine, v-HSVgDlNY constructed from the same parental vaccinia strain (v-NY) as

  v-env5NY. All animals were given a second dose of

  the same dose 8 weeks after the first immunization

  tion. Serum samples were collected twice weekly after immunization and assayed by ELISA and Western coagulation for antibodies specific for LAV / HTLV III. All animals exhibited self-healing of skin lesions, typical of vaccinia virus infection, and had normal physiological outcomes throughout the course.

experience.

  The methods for an ELISA type examination on chimpanzee sera were identical to those used for macaque sera. The results presented in Table V indicate that both experimental animals (124 and 149) underwent seroconversion after 8 weeks after first immunization and that antibody levels continued

  to increase after the second immunization injection. The ani-

  poor control (134) remained seronegative.

Table IV

  ELISA assay of serum samples from chimpanzees immunized with recombinant vaccinia viruses ELISA reading for serum antibodies specific for LAV / HTLV III Collection time Chimpanzee n 134 vHSVgDINY Chimpanzee n 124 v-env5NY Chimpanzee n 149 v-env5NY Before bleeding 8 weeks after the first immunization injection 2 weeks after the second immunization injection

0.084 + 0.015

0.085 + 0.010

0.075 + 0.012

0.100 + 0.005

0.185 + 0.020

0.237 + 0.017

0.123 + 0.025

0.403 + 0.027

0.523 + 0.073

ro N o -J

  To show that antibodies specific to LAV / -

  HTLV III detected in immunized animals were directed against envelope glycoproteins, the

  same sera by Western coagulation. The operating mode

  The field used was optimized for the detection of gp41 antibody, and it was 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 glycoproteins of

  veloppe, and that the level of these antibodies has increased

  after the second immunization injection.

  IMMUNOLOGICAL RESPONSES, MEDIATED BY CELLS,

  IN CHIMPANZES IMMUNIZED BY V-ENV5NY

  Peripheral blood lymphocytes, isolated from the same animals as those described above, were used.

  to show immunological responses, mediated by

  in these animals. Lymphocytes were isolated from

  peripheral blood from heparinized blood, by centrifugation

  ficolli-hypaque fugation 4 weeks after inoculation

  initial intradermal concentration of recombinant vaccinia viruses.

  These lymphocytes were seeded at 1 x 105 cells per well in 96-well microtiter plates in RPMI 1640 medium supplemented with 10% normal heat-inactivated human serum and penicillin /

  streptomycin. The glyco-

  LAV / HTLV III proteins (5, ug / ml) or glycoproteins

  envelope of LAV / HTLV III (1 μg / ml), isolated by chromato-

  lectin on purified LAV / HTLV III. Six days later, scintillation counting was determined by

  liquid medium, H3-thymidine incorporated into the cells.

  As shown in Table V, lymphocytes from both animals (n S 124 and 149) immunized with v-env5NY, proliferated in response to stimulation by the LAV / HTLV III virion or purified envelope proteins (approx. ). In contrast, animal 134, which has received vaccinia, recombinant herpes simplex v-HSgD1NY, has not

  product of lymphocytes recognizing LAV / HTLV III.

TABLE V

  Immunological cell-mediated response in chimpanzees vaccinated with 3H-thymidine vaccinia recombinants incorporated into Chimpanzee-stimulated lymphocytes n No immunization LAV / HTLV III antigen 124 v-env5NY 2842 37 132 26 370 149 v env5NY 375 20 292 27 115 134 v-HSVgD1NY 367 1 987 367 64 NA 452 567 222 72 NA 737 2,417,905 * peripheral blood lymphocytes Peripheral blood lymphocytes from

  all chimpanzees had high titres of

  poration of 3H-thymidine (38,870 to 109,790 cpm) after stimulation

  phytohemagglutinin (PHA, 2, ug / ml) and high levels of 3H-thymidine incorporation (42,172 at 12,067 cpm) after stimulation with blood lymphocytes

  xenogeneic human peripheral, grouped, having undergone irrational

  X-ray diagnoses. Peripheral blood lymphocytes

  from chimpanzees immunized with v-env5 and also the chimpanzee immunized with v-HSVgDlNY, showed strong proliferative responses to vaccinia viruses, then

  peripheral blood lymphocytes from chemotherapy

  non-immune panzés did not proliferate in response to a

  stimulation with vaccinia virus.

  Overall, the results shown in Table I of the main patent, on Tables I to V and Figures 11 to 13, indicate that (a) recombinant vaccinia virus v-env5 and its counterpart in the vaccine> v-env5NY, were able to elicit LAV / HTLV III specific humoral and cell-mediated immunologic responses in two subhuman primate species, macaques and chimpanzees, and (b) that recombinant viruses do not have not suppressed immunological responses & mediated by cells or

T cells, in vitro or in vivo.

  DECREASING THE NEUROTOXICITY OF RECOMBINANTS BUILT

  WITH THE V-NY STRAIN OF VACCINE VIRUS

  Groups of five mice (ICR strain) were injected into the brain containing aliquots of virus, containing x105 plaque forming units in 25-50 μl. The reported mortality ratio was reported 10 days later.

inoculation.

TABLE VI

  Mortality of cerebral mice by recombinants

inoculation

  vaccinia Inoculation v-NY v-env5NY v-env2NY; v-epv7NY smallpox vaccine (Wyeth) v-env5 v-env2 v-env7 Animals dead / animals treated by injection 0/5 0/5 0/5 0/5 3/5 / 5/5/5 saline solution 0/5 The results presented in Table VI indicate

  that vaccinia virus from a tissue culture

  plaque-purified plaque (v-NY strain) and its derivatives v-env5NY, v-env2NY and v-env7NY, have reduced neurotoxicity in comparison with strain WR and its derivatives. Since the strain of the New York City Board of Health

  health of New York City) served as an anti-vario-

  However, recombinants based on this strain should be better suited for the development of a vaccine for human use.

  EXAMPLE 2- RECOMBINANTS GAG OF BACULOVIRUS

  In the following example, we have built various

  Plasmid vectors containing chimeric genes comprising LAV / HTLV III gag coding sequences located downstream from AcNPV transcriptional control sequences.

  These chimeric genes, containing the promoter, were inserted

  AcNPV polyhedrin and the gag coding sequence of LAV / HTLV III, in the AcNPV genome, by in vivo recombination. Such recombinant viruses were identified and purified, and virus stocks were prepared from infected tissue culture cells. GAV-related immunoreactive proteins of LAV / HTLV III have been shown to be produced in vitro by the AcNPV recombinant. We

  has shown that cells infected with recombinant viruses

  have a positive immunofluorescence. Finally, recombinant AcNPV-infected cell lysates were positive in the ELISA assay when assays were performed with serum from patients with

  AIDS. A detailed description of each step of this

  embodiment of the invention is presented hereinafter.

GENERAL OPERATING MODES

  Cells and Virus Spodoptera frugiperda cells, clone Sf9, were obtained from the American Type Culture Collection (ATCC n CRL 1711) and propagated in Grace's Antheraea (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 cultured at 28 ° 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). purified this virus by plate on Sf9 cells containing 1% agar and

  0.8 x TNM-FH. The dilutions of virus stocks have been

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 the E. coli DNA polymerization was used at 200 units / ml in mM Tris-HCl (pH 7.4), 6 mM MgCl 2, 10 mM 2-mercaptoethanol.

37 C for 30 minutes.

  Viral DNA for transfection was prepared from wild-type non-occluded virus (VNO) stocks using the following Miller procedure.

  L.K; Miller D.W. and Safer, P .: we obtained a pellet of

  trifugation of the VNO particles to separate them from the supernatant liquid through a pad of 25% sucrose, mM NaCl and 10 mM EDTA, by centrifugation at 90,000g for 1 hour at 5 ° C. The supernatant liquid and the The virus pellet was resuspended in 0.5 ml of dye-lysate buffer (10 μM Tris, pH 7.6, 10 mM 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 C with occasional agitation.

  The DNA was extracted with an equal volume of phenol: chlorinated

  form: isoamyl alcohol (25: 24: 1). The DNA was then precipitated at 20 ° 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 ramis suspended.

  in TE (10 mM Tris, 1 mM EDTA, pH 7.5) before use.

  read for transfection or restriction enzyme analysis. Plasmid DNA was prepared using the procedure of Summers, M.D. and Smith, G.E. as follows: A single colony of bacterial cells, from a freshly prepared plate, was inoculated into 1 ml of

  Luria broth (BL) supplemented with a suitable antibiotic.

  After incubation at 37 ° C. for 12 to 18 hours,

  ml of culture in 200 ml of Luria broth plus anti-

  biotics in a 500 to 1000 ml flask, and incubated in a shaking incubator (37 C) overnight (12 to 18 hours). The cultures, of 200 ml each, were transferred to centrifuge bottles, and

  centrifuged at 2500 rpm for 10 minutes. After removing

  supernatant fluid, each suspension was resuspended

  cell pellet in 30 ml of STET buffer (8% saccharine

  rose, 0.5% Triton X-100, 50 mM EDTA (pH 8.0), 10 mM Tris-Cl, pH 8.0) at room temperature and was transferred to a 125 ml flask. ml. Then 3 ml of 10 mg / ml of lysozyme (freshly prepared in STET buffer) was added to each flask, and each flask was swirled to mix, followed by incubation for about 5 minutes. at room temperature. Each flask (approximately 1 every 5 seconds) was then gently swirled directly over a flame until the cells began to clot and turn white. As bubbles formed, the flasks were transferred to a boiling water bath for seconds, after which the flasks were cooled in an ice-water bath for 2 minutes or more. We paid

  slowly viscous mixtures in centrifuge tubes.

  50 ml round bottom runners, and centrifuged for minutes at 16,000 rpm using a SW 28 rotor (Beckman, CA) in a centrifuge for preparation. The supernatant fluid from each tube was carefully poured into a disposable polypropylene centrifuge tube, to which a volume of isopropanol or two volumes of ethanol was added, and the DNA was precipitated. -80 C for 20 minutes (or -20 C for 2 hours or more),

  and centrifuged at 2500 g for 15 minutes or more.

  After removal of the alcohol, 2.5 ml of extraction buffer (per liter: 12.1 g of Tris, 33.6 g of Na 2 EDTA, 2H 2 O, 14.9 g of KCl, pH 7.5) was added. on the base, which we then

  swirls to loosen the lysate from the bottom of the tube.

  100 μl of 10 mg / ml of RNase A (dissolved in 0.1X TE and pretreated by 10 minutes of boiling) were added and incubated for 30 minutes at 37 ° C. 200 ug Proteinase K and then incubated at 40-50 ° C. for about 30 minutes, after which 250 ul of 10% Sarkosyl was added and incubation continued for 3 hours. at least. In order to prepare CsCl gradients, 80 μl of 10 mg / ml ethidium bromide per ml of DNA solution was added to each tube. 1.04 g of CsCl was then added to each tube.

  ml of DNA solution and gently

  to dissolve. After transfer to "Quick Seal" tubes (R), the gradients were centrifuged to equilibrium at 55,000 rpm for 16 hours in a 70.1 Ti fixed angle rotor, resulting in two bands visibly well separated from each other. DNAs in which the upper band comprises linear DNA and notched while the lower band comprises covalently closed circular DNA. The lower band (covalently closed circular DNA) was collected and

  poured into a 15 ml centrifuge tube made of polypropylene

  lene, in which water was added to bring the volume to about 6 ml in each tube. Ethidium bromide was extracted from the DNA using an equal volume of alcohol

  isoamyl, and the pink phase was removed and discarded.

  higher). Extraction was repeated until the upper phase was colorless, and the lower phase (DNA) was clear and colorless. About 5 ml of DNA solution should remain in each tube (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 ° C overnight or at -80 ° C for 10 minutes, and a pellet was formed.

  by centrifugation at 2500 g for 20 minutes. After

  all the alcohol was added to each pellet

  500 μl of 0.1X TE, and the pellet was again resuspended.

  pension at 65 C for 10 minutes at least. We can keep

DNA at 4 C.

  Plasmid DNA was also prepared as described in Maniatis et al., 1982, Molecular Cloning, Cold Spring Harbor Laboratory (page 8696). DNA ligase was purchased from New England Biolabs, and used at 10,000 units / ml in 50 mM Tris-HCl (pH 7.8), 10 mM MgCl 2, 20 mM DTT, 1 mM ATP. DNA analysis on agar gels was as described in Maniatis et al., 1982, Molecular Cloning,

Cold Spring Harbor Laboratory.

  CONSTRUCTION OF PLASMID VECTORS CONTAINING PROMOTER

  LIGATION-RELATED ACNPV TO LAV / HTLV III GAG GENE ENCODING SEQUENCES (pAc-gag1) The construction and plasmid vectors containing coding sequences of the LAV gene are described below.

  HTLV III gag, which is preceded by sequences of com-

  transcript of AcNPV and followed by poly-

* hedrin. These recombinant plasmid vectors were then used to insert the coding sequences of LAV / HTLV III gag into

  the baculovirus genome by in vivo recombination.

  The coding sequence of LAV / HTLV III gag (FIG. 14) was purified from pKS-5, a J19 lambda subclone (Wain-Hobson, S, et al., 1985, Cell 40: 9) and we have it

  inserted into the plasmid Ac610 downstream relative to the promoter

  baculovirus polyhedrin contained in pAc610 to construct pAc-gag1 (Figure 15). In this construct, the LAV / HTLV III specific nucleotide sequence is preceded by the gene forming polyhedrin promoter.

  chimera followed by the sequence of the pdlyedrine gene.

  As previously explained, pKS-5 consists of a 3148 base pair fragment SStI to KpnI of the inserted portion of LAV / HTLV III DNA contained in lambda J19 cloned into the SstI and KpnI site of pUc18. It was obtained by cloning the J19 SstI restriction fragment into the pUC18 SStI site to create pBT-1, which was then digested with KpnI and re-ligated. This DNA was used to transform E. coli, and plasmid pKS-5 was isolated. The specific DNA of LAV /

  HTLV III contained in pKS-5 goes from the SstI site housed in the nucleus

  # 224 to the KpnI site at nucleotide 3372 on the LAV / HTLV III genome (Wain-Hobson, S et al., 1985, Cell 40: 9;

see also Figure 14).

  10 μg of pKS-5 was digested to completion with HincII and SStI, and the resulting fragments resolved on a low melting 1% agar gel. The fragment containing 1818 base pairs (about 0.20 μg) was excised from gel. This fragment contained specific sequences of LAV / HTLV III ranging from nucleotide # 224 to # 2042, as shown in FIG. 14. It contains

  the coding region for the entire gag gene.

  Five micrograms of pAc610 were digested until completion with SmaI and SstI, and fractionation was performed on a 1% low melting agar gel; the DNA band was excised from the gel (approximately

0.4 μg) (Figure 15).

  Both gel slices were melted at 65 ° C. for 10 minutes and 3 μl of LAV / HTLV III specific fragment containing 1818 was ligated.

  pairs (digestion with HincII and SstI) on a micro-

  1 pAc 610 (digestion with SmaI and SstI) in a total volume of 40 μl. Incubation took place at 23 ° C (room temperature) for 16 hours. The ligation mixture was then heated for 10 minutes at 65 ° C and used to transform E. coli strain HB101. Plasmid DNA from transformants capable of resisting

  ampicillin, to detect the presence of the insulin

  corresponding to LAV / HTLV III. The structure of this plasm

  mide, pAc-gag1, is shown in FIG.

  CONSTRUCTION OF RECOMBINANT BACULOVIRUS CONTAINING GENE

LAV / HTLV IOI GAG CHIMER (Ac-gag1) AcNPV plates were purified and

  killed the spread on Sf9 cells. Wild-type AcNPV DNA was isolated and pAc-gagl was purified as

  previously described (see above).

  The insertion of the chimeric sequences was carried out

  of LAV / HTLV III gag in the genome of AcNPV, by recombinant

25877'20

  In vivo generation made possible by the fact that the gag sequences of pAcgag1 are surrounded by coding AcNPV sequences. Co-transfection of plasmid pAc-gagl DNA

  with wild-type AcNPV DNA allowed for a recom-

  occur between the polyhedrin sequences on the plasmid and homologous sequences of the AcNPV genome. The insertion of the chimeric gene occurs as a result of double recombinations in the surrounding or flanking sequences. Such recombinants will have the chimeric gene inserted

  in the polyhedrin gene of AcNPV and, therefore, will be

  to produce occlusion bodies. Recombinant plates can be selected by visual examination

  to detect the absence of occlusion virus.

  1 g of AcNPV DNA, 5 μg of pAc-gag1, and 15 μg of calf thymus DNA were mixed, and precipitated with ethanol. The precipitate was washed with% ethanol, allowed to air dry under a hood for culture.

  of tissues, and resuspended in 437.1 H2O.

  CaCl2 was added up to 0.25 M (63 μl of 2M CaCl 2 for 437 μl of DNA in H 2 O) while bubbling air through it.

  in 500 μl of 2XHEBS the solution was added dropwise.

  DNA (1 drop / 4 seconds). 2XHEBS contains, per ml, 16 mg of NaCl, 2 mg of D-glucose, 0.75 mg of KC1, 0.5 mg of Na2HPO4.12H2O, 9.5 mg pH 7.1. The mixture was incubated at room temperature for 30 minutes and added to a 60 mm tissue culture dish.

  containing 3 x 106 Sf9 cells in 2 ml of insect

  of Grace containing 10% fetal calf serum and

  antibiotics (no yeast hydrolyzate or hydrolyzate

  sat lactalbumin). The solution was added dropwise

  The cells were incubated with 4 ml of Grace + FBS + antibiotics and incubated for 90 minutes. with Grace's middle + FBS +

  antibiotics + 20% dimethylsulfoxide.

  The plate was rinsed 3 times with complete medium and incubated at 28 ° C. with 4 ml of complete medium. After 5 days, the supernatant was collected and clarified at 1000 g for 5 minutes. The virus stock was titrated on Sf9 cells and plaques without occlusion were visually identified. Plates were removed without occlusion and two more plate treatments were performed. These plates were expanded on Sf9 cells and virus stocks were prepared and used for characterization.

  subsequent. A schematic representation of the construction

  The recombinant is shown in Figure 16.

  EXPRESSION OF LAV / HTLV III GAG RELATED PROTEIN

  IN CELLS OF TISSUE CULTURES INFECTED BY

RECOMBINANT BACULOVIRUS

  Recombinant AcNPV carrying the LAV / HTLV III gag chimeric gene has been shown to be capable of expressing, after infection of the cells in tissue culture, RNA and related proteins to LAV / HTLV III gag. It has also been found that these proteins are immunoreactive with serum

  from AIDS patients.

  The identification of LAV / HTLV III gag specific RNA in Ac-gag1 infected cells Ten boxes of 100 ml of Sf9 cells (approximately 107 cells / dish) were infected with wild-type AcPNV or

  its recombinant, Ac-gagl, at a multiplicity rate of infection.

  4. 24 hours after infection, the cells were harvested and washed twice in 0.15 M NaCl, 0.01 M Tris-HCl, pH 7.2. Polyadenylated RNA was isolated,

  as described (Purchio, A.F. and Fareed, G.C, 1979, J. Virol.

  29: 763-769). The RNA was fractionated on a 1% gel of agarformaldehyde (H. Lehrach et al., 1977, Biochemistry 16: 4743-4251), transferred to a "nylon" filter.

  ("Hybond") and irradiated with ultraviolet light.

  Filter hybridization was induced on 3 P-labeled pKS-5 DNA in 0.9 M NaCl, 50 mM sodium phosphate (pH 7.0), 5 mM EDTA, 0.1% SDS, 4 XDenhardts, RNAt 0.4 mg / ml, calf thymus DNA -0.25 mqiml, 50% formamide for 16 hours at 42 C. Filters were washed four times in SDS, 0.1%, 0.25XSSC during 30 minutes at 65 C and exposed to a film "Cronex 4" for X-rays, using intensifying screens "Lightening Plus". Figure 17 shows that a 2.2-kb major band is detected in cells infected with recombinant AcNPV,

  but not in cells infected with a wild type.

  IDENTIFICATION OF PROTEINS RELATED TO LAV / HTLV III GAG

  EXPRESSED IN CELLS INFECTED WITH BACULOVIRUS

  RECOMBINANT, USING IMMUNOPRECI-

PITATION

  The immunoprecipitation assay, described in

  below, shows that cells infected with baculo-

  recombinant virus of the present invention-synthesize proteins that are specifically immunoreactive with

  serum from AIDS patients.

  Sf9 cells were seeded on ml dishes (5x10 6 cells / bunch) and infected with wild-type AcNPV in Ac-gag1. At 24, 48 and 72 hours after infection, the media were replaced with methionine-free media, and the cells were incubated for 30 minutes.

  minutes at 28 C. At the end of this time, we replaced the

  places with methionine-free media containing 100 iCi / ml

  of methionine and the labeling was allowed to proceed.

follow for 2 hours at 28 C.

  At the end of the labeling period, the cells were washed twice with 0.01M Tris-HCl (pH 7.2), 0.15M NaCl.

  and collected by centrifugation. We have reinstated

  pension cell pellets, from each boot

  60 mm, in 1 ml of lysis buffer: 1% NP-40 (poly-

  oxyethylene (9) p-tert-octylphenol), 0.5% sodium deoxycholate, 0.1M NaCl, 0.01M Tris-HCl (pH 7.4), 1 mM EDTA, and clarified the lysate by centrifugation

for 30 minutes at 100,000 g.

  Immunoprecipitation was performed by adding 4.1 of heat-inactivated human serum from a control or AIDS patient population.

  to 500 ml of cell lysate. After half an hour of incu-

  at 4 ° C, 80 μl of activated Staphylococcus aureus cells ("Pansorbin" cells, Calbiochem-Behring Corp., La Jolla, CA, USA) were added and

  incubation to continue for an additional half-hour

  The immunoprecipitation supplements were collected by centrifugation for 5 minutes at 5000 rpm. in a Sorvall A384 rotor at 4 ° C., and washed once in 1M NaCl + 0.1% NP-40 + 0.01 M Tris-HCl, pH 7.4, and three times in RIPA buffer ( 10 mM Tris-HCl, pH 7.2, 0.15 M NaCl, 1% sodium deoxycholate, 1% lauryl sulfate

  of sodium, 1% of "Triton X-100"). The immune

  The precipitates thus washed, suspended in 80 μl of Laemmli sample buffer, were boiled for 1 minute and centrifuged for 5 minutes at 5000 rpm. in a Sorvall A384 rotor. The immunoprecipitated proteins present in the supernatant were analyzed by electrophoresis on 10% SDS polyacrylamide gels. After electrophoresis, the gels were stained with blue dye

  Coomassie was treated with sodium salicylate (30 minutes

  at room temperature in sodium salicylate

  1M) and dried for fluorography.

  It has been suggested that mature gag proteins (34,000 daltons, p24, 16,000 daltons, p16, 14,000 daltons,

  p14) are obtained from a larger protein pre-

  slider of 55,000 daltons. Figure 14 shows that proteins with molecular weights of 67,000 daltons, 10,000 daltons, 40,000 daltons, 34,000 daltons, 32,000 daltons and 24,000 daltons have been specifically immunoprecipitated

  with serum from AIDS patients.

  In order to determine whether there is a relationship

  precursor shell / product among immunoreactive proteins

  In any case described in Figure 18, hunting pulse experiments were performed. 24 hours after infection, cells were labeled for 5 minutes with [5Si methionine; the cells were then washed with complete medium and incubated with complete medium at 2, 4 and 8 hours. The cells were then harvested and immunoprecipitated. The fractionally immunoprecipitated proteins were subjected to gels

  polyacrylamide 10% SDS, as described above.

  Figure 19 shows a fluorogram of this experiment.

  The data suggest that p67, p55, p40, p34 and p32 incorporate the marker after 5 minutes. After an 8 hour hunt, the marker decreases in p67 and p55 and increases

  in p34. The amount of radiolabel in p40 remains relative to

constantly constant.

  ELISA IDENTIFICATION OF LAV-RELATED PROTEINS /

HTLV III GAG

  Ac-gagl was infected with ten 100-ml

  each holding 10 Sf9 cells. 24 hours after infection, the cells were harvested, washed twice in 0.01M Tris-HCl (pH 7.2), 0.15M NaCl and frozen and thawed three times. The cells were disrupted with 2 ml of phosphate buffered saline containing 0.5% NP-40 on ice for 5 minutes. The lysates were centrifuged at 1000 x g

  for 10 minutes and the supernatant liquid was removed.

  4 ml of carbonate buffer (50 mM sodium carbonate, pH 9.6) was added and the mixture was rotated in an Eppendorf centrifuge for 20 minutes. We removed

  the supernatant liquid and diluted in

  as shown in Table III. These lysates were used to coat microtiter plates with 96 wells (50 μl / well) overnight at 4 ° C. 300 μl of a blocking reagent (5% non-fat milk powder, 0.01% thimerosol, 0.1% antifoam A in phosphate buffered saline) was added, and the reaction mixture was added to the reaction medium. 'were incubated during

  minutes at room temperature. We removed by vacuum

  The blocking reagent was added and 50 μl of a 1: 100 dilution of human serum in blocking reagent was added to each well. Prior to addition to the troughs, the diluted sera (sera from patients with AIDS or normal human serum) were incubated at 37 ° C for 45 minutes. Each hollow was allowed to react & 37 C during 1

  hour. The hollows were then washed three times with chlorine

  sodium chloride + "Tween 20" (polyoxyethylene sorbitan monolaurate). 100 μl of goat anti-human horseradish peroxidase conjugate (1: 100 dilution in goat normal serum containing 0.01%) was added to each well.

  of thimerosol) and allowed to react for 1 hour at 37 ° C.

  The plates were washed and 100 μl / well

  buffered tread plus chromogenic reagent: the

  poned is hydrogen peroxide, citric acid and

  phosphate buffer; the chromogenic reagent is the tetra-

  methylbenzidine in dimethylsulfoxide. We have incubated

  plates for 30 minutes at room temperature.

  The reaction was stopped by adding 100 μl / well of 3N sulfuric acid. The optical absorptance of the mixture was measured at 400 nm with a reference absorbance at 630 nm.

  reaction. The results are presented in Table VIII.

  TR1, Y-1, CF22C and CF9 correspond to serum from AIDS patients; 2, 16, 21 and 48 are serum

normal human.

TABLE VII

  ABSORPTION VALUES DURING AN ASSAY

  Antiserum Dilution of lysate -1 AIDS TR1 Y-1 2,619 2,504 2,502 2,305 2, 486 2,319 2,303 2,209 CF22C CF9

NORMAL SERUM:

  0.221 0.229 0.176 0.165 0.193 0.205 0.200 0.249 -2.471 2.479 2.346 2.306 2.317 2.241 2.111 2.012 È, 204 0.187 0.175 0.135 0.190 0.176 0.185 0.169

1,400-1

  2,235 2,216 2,046 2,006 1,924 1,838 1,339 1,305

0 -312

0.176 0.217 0.139 0.216 0.170 0.217

0.163,

  2,076 2,119 1,564 1,595 1,393 1,331 0.855 0.850 0.199 0.227 0.131 0.210 0, 189

0, 199

0.177 0.175

"EL I SA"

Ac-gag 1

1000 1

1,499 1,500 1,014 1,078

0.8 11

0.848 0.512 0.455

800- 1

  1,786 1,877 1,298 1,282 1,009 1,068 0,636 0,659 0,228 0,208 0.144 0.175 0, 237 0.197 0.190 0.173 CD -4 0.222 0.210 0.129 0.145 0.165 0.224 0.134 0, 143 r% 3 oo Po on uA.I 0c 1- 'Overall, The results shown in Figures 17-19 and Table VII indicate that the recombinant baculoviruses described herein were capable of (a) expressing the inserted LAV / HTLV III gag gene; (b) modify or treat such gene products; and (c)

  produce specifically immunogenic antigenic

  reactive with serum from AIDS patients.

EXAMPLE: RECOMBINANT GAG-VACCINE

  In the following example, we constructed

  plasmids which contained complex chimeric genes

  taking LAV / HTLV III gag coding sequences located downstream from the vaccinia virus transcription control regions. The chimeric gene containing vaccinia 7.5 K promoter virus and the LAV / HTLV sequence was inserted.

  III gag in the genome of vaccinia virus, by recombinant

  in vivo. Such recombinant viruses were identified and purified, and virus stocks were prepared from cells obtained by culturing on infected tissue. It has been shown that immunoreactive proteins related to LAV / HTLV III gag are produced in vitro by

  recombinant vaccinia. 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

  about 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 have been constructed which contain

  The 7.5K promoter of vaccinia virus linked by ligand was

  different lengths of the LAV / HTLV III genome

  gag coding sequences. The source of the sequences

  coding of LAV / HTLV III gag were two subclones

  lambda J19 (Wain-Hobson et al., Cell 40: 1985), pKS- and pSS-5. Plasmid pKS-5 contains a 3148 base pair fragment ranging from SStI to KpnI (nucleotide # 224 to # 3372) of the LAV / HTLV III DNA inserted at the sites.

  corresponding to pUC18. Plasmid pSS-5 contains a fragment

  5107 base pairs of SstI to SalI (nucleotide # 224

  to 5221) of the LAV / HTLV III DNA inserted at the corresponding sites.

  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 unique EcoRI site downstream of the unique SmaI site (see figure). The LAV / HTLV III specific sequences, inserted in pGS62, all begin with a ThaI site (FnuDII) located 76 base pairs upstream of the gag gene initiation codon. Plasmids pv-gag1, pv-gag2, pv-gag3, pv-gag4 and pv-gag5 contain specific LAV / HTLV III sequences from this ThaI site to the EcoRI (at 4194) and KpnI (at 3372) sites. , EcoRv (at 2523), BclI (at 1975) or BglII (at

  1642), respectively. The description of the construction

  of each plasmid, performed below, is facilitated when

reference is made to FIG.

  Construction of pv-gagl: 5 μg of plasmid pSS-5 DNA was digested to completion with ThaI EcoRI restriction enzymes. The resulting fragments were

  resolved on a 1% LMP agar gel. A fragment has been isolated

  3.9 kbp, containing the coding sequences of LAV / HTLV III gag and ligated to DNA of

  pGS62 previously subjected to digestion with SmaI and EcoRI.

  The ligation mixture was used to transform E. coli HB101 and clones capable of withstanding

  ampicillin. Plasmids from colony

  individual tests to determine the presence

  inserted fragment of 3.9 kilopytes of base (kbp), per

  restriction lysis and regeneration of the EcoRI site

  at the ligation junction. The resulting construction con-

  holds the entire coding sequence of the gag genes

  and protease (prt), and a major part of the

  open pol, up to the EcoRI site at nucleotide 4194.

  Construction of pv-gag2: 5 μg of plasmid pKS-5 was digested to 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 inserted sequence from LAV / HTLV III and the cloning sites

  multiples of plasmid pUC18. The resulting fragments were solved

  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 performed on pGS DNA. 62, previously subjected to SmaI digestion and treated with CIAP. The ligation mixture was used to transform E. coli HB101 and clones capable of

  to resist ampicillin. Plasmids,

  from single colonies to tests for the presence of the 3.1 kilobase base fragment, and the orientation of the insertion was determined by restriction analysis. The resulting construct pvgag2, contains the entire gag gene, the gene prt and part of the frame

  open reading pol 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 insertion at the site. SmaI of pGS62. The final construct, pv-gag3, contains all the gag and ready genes and a small portion of pol fused on the 3 'portion of the TK gene.

of vaccinia virus.

  Construction of pv-gag4: 5 μg of pSS-5 plasmid DNA was digested to completion with SStI and BclI. The resulting fragment, containing the sequence corresponding to LAV / HTLV III gag, was purified. This fragment was ligated to a plasmid vector 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 at the EcoRI site in this multiple site cloning vector. Digestion of this intermediate construct with the restriction enzymes ThaI and EcoRI generated a

  a fragment of 1.72 kbp that could be easily

  insert 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 fused prt gene.

  on the 3 'part of the TK gene of vaccinia virus.

  The construction of plasmid pv-gag5 used the same two-step strategy as for pv-gag4. 5 μg of pSS-5 were digested with SstI and BglII restriction enzymes. A 1.42 kbp fragment, containing the sequence corresponding to LAV / HTLV III gag, was isolated and this fragment was purified on a 1% LMP agar and inserted into the plasmid pIC19R. SstI and BglII sites. Ligation of the gag sequence to the BglII site on its corresponding site in pIC19R allows (a) formation of a stop codon in

  phase with the open reading frame gag, and (b) the juxtaposition

  position of the BglII site at the adjacent EcoRI site on the multi-site cloning plasmid. This intermediate construct was then digested with ThaI and EcoRI. A 1.39 kp fragment containing the sequences corresponding to LAV / HTLV III gag was purified and this fragment was ligated to the SmaI and EcoRI sites on the pGS62 plasmid. The resulting plasmid, pv-gagS, contains most, but not all, of the gag coding sequence (up to the BglII site) fixed by ligation on a translational stop sequence in the

frame.

  Construction of recombinant vaccinia viruses containing LAV / HTLV III gag chimeric genes Insertion of LAV / HTLV III gag chimeric genes from pv-gag1, pv-gag2, pv-gag3, pv-gag4 and pvgag5 vector plasmids genome of vaccinia virus, was performed by the same protocol as that used for the construction of v-env5 and v-env2 (see above). In the following examples, all recombinant viruses were constructed using the plaque-purified strain from the New York City Board of Health.

  the health of New York City) (v-NY, see above).

  TK recombinants were selected and plaque purified three times before expanding into virus stocks which were used for subsequent characterizations. Recombinant viruses from pv-gag1, pv-gag2, pv-gag3, pv-gag4 and pv-gag5 were designated v-gaglNY, 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 Recombinant vaccinia viruses carrying the LAV / HTLV III gag chimeric gene, described above, are capable of expressing related proteins to LAV / HTLV III gag, when introduced by infection into

  tissue culture. Some of these recombinants have been

  to pass the protein corresponding to precarious gag

  p55, in the state of the mature proteins p25 and p18. These proteins have been found to be immunoreactive with serum of AIDS patients and / or with antibodies

  monoclonal against gag p25 or p18 proteins.

  At an infection rate of 10,

  BSC-40 confluent flasks, in a 60 mm dish, with parental-type vaccinia virus (v-NY) or its recombinant derivatives, v-gaglNY, vgag2NY, 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 buffered saline.

  by phosphates and collected by centrifugation.

  The centrifugation pellets of infected cells were

  suspended in 0.3 ml of a sample buffer

  of Laemmli and the lysis was performed by 4 minutes of boiling

  tion. The entire cell protein was resolved in μl of cell lysate by electrophoresis on a 15% SDS-polyacrylamide gel. As a control, a sample of purified virion LAV / HTLV III and an aliquot of a lysate of cells infected with elements were included.

non-infectious ("mock").

  The contents of the gel were electrophoretically transferred to a sheet of a nitrocellulose filter. The filter was first reacted with patient serum

  with AIDS, or with a combination of mono-

  clones against p25 and p18 gag proteins. After extensive washings, the filter was then reacted with alkaline phosphatase conjugated goat anti-human immunoglobulin (AP conjugate) antibodies in cases where patient serum was used. or with

  goat anti-mouse immunoglobulin antibodies to

  AP judgment, 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

  washes as described above with regard to the identification of

  related proteins in the LAV envelope /

  HTLV III. The final wash after the second reaction of

  body was carried out in 0.1M Tris (pH 9.5) with 0.1M NaCl and 5mM MgCl2. Antibody-bound antibody was detected on the filter by reacting the filter with a solution containing 0.1M Tris (pH 9.5), 0.1M NaCl, mM MgCl2, 0.33mg / ml. ml of bromo-chloroindo phosphate

  and 0.17 mg / ml of nitrotetrazolium buffer. After reaction

  filtering with the chromogens, the filters were washed

  with water, they were air dried and photographed.

  The results of this analysis are presented on

  Figure 21. The recombinants v-gagiNY and v-gag2NY contain

  all the gag and ready genes. These recombinants express a family of LAV / HTLV III related proteins that co-migrated with the major gag proteins of LAV /

  HTLV III, p55, p40, p25 and p18. These proteins have been immuno-

  reactive with both AIDS patient serum (Figure 21B) and mouse monoclonal antibodies against p25 and p18 (Figure 21A). The recombinant v-gag3NY contains the gag and prt genes, but its open reading frame pol is ligated in phase with the carboxy terminus of the vaccinia TK gene. V-gag3NY was also able to express the primary gag gene product, p55. However, the treatment of p55 seems to have been less effective than in the case of vgaglNY and v-gag2NY, since there was much less p25 and p18, compared to p55,

  in lysates of cells infected with v-gag3NY. The recom-

  hog v-gag4NY contains the entire gag gene, but only

  part of the gene ready. He synthesized a similar LAV / HTLV III related protein in p55 dimensions. The recombinant v-gag5NY contains only a portion of the gag gene and expressed a truncated 43 kD protein (Figure 21C). Although v-gag4NY and v-gag5NY do not seem to have

  effectively treated their gag-like proteins.

  Mayor, their products were immunoreactive with monoclonal antibodies against p25 and p18. In sum, despite

  differences in treatment capacity, the five recom-

  binants have all been able to express immune

  which contained the major epitopes of

core ines of LAV / HTLV III. -

  EXAMPLE: RECOMBINANTS OF BACULOVIRUS ENVELOPE

  In the following example, a vector plasmid containing a chimeric gene comprising the LAV / HTLV III envelope coding sequences housed downstream by

* report to transcription control sequences of AcNPV. The chimeric gene contained the AcNPV polyhedrin promoter and the sequence

  LAV / HTLV III env coding was inserted, by in vivo recombination, into the AcNPV genome. The recombinant virus has been identified and purified, and

  virus stocks from infected supernatant cells

  Tees. It has been shown that LAV / HTLV III env-related immunoreactive protein is produced in vitro

  by the recombinant baculovirus. A detailed description

  of each step of this embodiment of the invention

  is presented below. The general operating procedures used

  for this embodiment are as described

  above for baculovirus recombinants.

  Construction of vector plasmids containing the AcNPV promoter linked by ligation to the coding sequences of the LAV / HTLV III env gene The coding (or coding) sequence of the LAV / HTLV III env gene was purified from pvenv5 (see above,

  in the part concerning the construction and characterization

  recombinant vaccinia virus containing the LAV / HTLV III envelope chimeric gene), which was used for the construction of recombinant vaccinia virus v-env5, which has been shown to express related proteins & approx. The env coding region in pv-env, as well as the untranslated sequences of 96 base pairs close to 5 'and 223 base pairs close to 3', were surrounded.

  at both ends by BamHI sites. A digestion par-

  of this plasmid, which also contained an internal BamHI site) using the BamHI restriction enzyme generated a 2.9 kbp fragment containing only

  specific sequences of LAV / HTLV III. We have resolved this frag-

  (about 0.5 μg) and purified from a 1% LMP agar, and ligated to 0.5 μg of the pAc610 vector plasmid, which had been pre-linearized with BamHI and treated with CIAP. The ligation mixture was used to transform the MC1000 strain

  of E coli and the colonies resistant to

  picilline. Plasmid DNA from individual transformants was tested for presence assays.

  sequences corresponding to LAV / HTLV III env (i.e.

  say the formation, by BamHI digestion, of the fragments of 2.3 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 named pAc-env5; Its structure

is shown in Figure 22.

  Construction of the recombinant baculovirus containing the LAV / HTLV III env chimeric gene The insertion of the LAV / HTLV III env chimeric gene into the AcNPV genome is carried out by recombination in vivo as explained above (construction of the baculovirus- recombinant containing the chimeric gene LAV / HTLV III gag). The introduction

  by transfection of 1 mg of AcNPV DNA, 5 mg of pAc-DNA

  env5 and 15 mg of calf thymus DNA in Sf9 cells was performed as described in the same paragraphs above. After 5 days of incubation at 28 ° C. with 4 ml of complete medium, the supernatants were collected and clarified by centrifugation at 1000 g for minutes. The virus stock was titrated on Sf9 cells, and the recombinant virus was first identified by the MD Summers and GE Smith plate hybridization assay: Cells were seeded Sf9 on 60 x 15 mm culture plates at a density of 2.5 x 10 6 cells per plate in serum-free TNM-FH medium. After fixing the cells, the medium was removed and 1 ml of a diluted virus inoculum was added to each plate. At the end of the hour of incubation at 27 ° C., all of the inoculum was removed from each plate and 4 ml of an upper LMP agar layer 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 colonies or plaques of

  the. The agar plates were then allowed to dry

  at night or longer, abandoning them in an environment

  unhumidified environment. When dry enough, the top agar layers were removed and a dry nitrocellulose filter was placed on top of it.

  the cells remaining in the culture plate. We have saturated

  with a solution A (0.05 M Tris-HCl, pH 7.4 and 0.15 M NaCl) a 47 mm circle of Whatman 3MM paper and this paper was placed over the nitrocellulose filter

  in the culture plate. After absorption, care was taken

  removed the nitrocellulose filter and placed it

  (the side of the cells on top) on a What-What-What-Is

  saturated with a solution a (0.5N NaOH, 1.5M NaCl).

  After 2 and 3 minutes, the nitro-filter was air-dried.

  cellulose on paper towels and transferred to Whatman filter paper saturated with a C solution (1. OM Tris-HCl, pH 7.4, 1.5 M NaCl). After 2 to 3 minutes, the nitrocellulose filter was air-dried on paper towels and immersed in a Petri dish filled with a D solution (0.3M). NaCl, 0.03 M sodium citrate). The filter was then removed and dried on paper towels, and heated at 80 ° C for 2 hours under vacuum. The 32 P-labeled pRS-3 filter was fixed by hybridization as a probe. Recombinant viruses were taken and titrated again on Sf9 cells. We identified,

  by visual examination, cell plates without occlusion.

  They were purified three more times and used

  to prepare virus stocks for further studies

  quent. Expression of LAV / HTLV III env-related proteins in infected tissue culture cells Dar of recombinant baculovirus Ac-env5 It has been shown that recombinant AcNPV, carrying the chimeric LAV / HTLV III env gene, is capable of expressing related proteins to LAV / HTLV III env, by infection of cells in a tissue culture. These proteins have been found to be immunoreactive with serum from

  AIDS patients as well as monoclonal antibodies

  clones that define glycoproteins gpl10 and gp41

  related to the envelope of LAV / HTLV III.

  Sf9 cells (1 x 107) seeded on a 100 mm dish were infected with wild-type AcNPV, or its Ac-env5 recombinant, at an infection index of 5. 28 hours after infection, replaced the medium with methionine-free medium not containing additional sera, and incubated for 30 minutes. After this period, the medium was replaced with 2 ml of a methionine-free medium containing 100 μCi / ml of E35SI methionine, and the labeling was allowed to continue for 2 hours at 28 ° C. At the end of the labeling period. cells were washed twice with phosphate buffered saline and resuspended in 1 ml of lysis buffer, as described above for the identification of

  proteins related to the envelope of LAV / HTLV III, ex-

  in cells infected with vaccinia virus

  recombinant, using immunoprecipitation techniques).

  Immunoprecipitation was performed as described in the above paragraphs using serum from AIDS patients as well as mouse monoclonal antibodies against gpl10 or gp41. The results of this assay, as shown in Figure 23, indicate that the recombinant Ac-env5 virus synthesized primarily a 150 kD protein, related to the LAV / HTLV III envelope. Since it comigrates with gp150 synthesized by vaccinia recombinant v-env5 and has been immunoreactive with AIDS patient serum as well as with monoclonal antibodies against gpl10 and gp41, this protein represents the glycosylated precursor.

  of the envelope protein gp150. Transformation treatment

  The formation of this molecule was not effective since gpl10 and gp41 were not detected during the 2 hour labeling period. However, since the major epitopes of gpl10 and gp41 are present on the precursor, this protein is potentially interesting as well.

  as a diagnostic reagent than as an immunogen.

DEPOSIT OF MICROORGANISMS

  The following strains of E. coli carrying the

  mides listed, were deposited in the service called "Agricul-

  Tural Research Culture Collection (NRRL), Peoria, IL (United States of America) and have received the following listing numbers: E. coli Strain Plasmid Registration Number K12 MC1000 pv-envl NRRL B-18003 K12 MC1000 pv NRRL B-18004 K12 MC1000 pv-env5 NRRL B-18005 K12 MC1000 pv-env7 NRRL B-18110 K12 HB101 pv-gagl NRRL B-18111 K12 HB101 pAc-gagl NRRL B-18105 K12 NF1829 pAc-env5 NRRL B- 18109 The following recombinant viruses were deposited

  American -T-Ype- Culture Collection, Rockville, MD, USA

  United States of America, and have received the following numbers of repository Recombinant virus Deposit number v-env2 ATCC VR 2114 v-env5 ATCC VR 2213 venv7 ATCC VR 2148 v-env5NY ATCC VR 2149 v-gaglNY ATCC VR 2150 Ac-gagl ATCC VR 2147 Ac-env5 ATCC VR 2151

  It should not be considered that the framework of the pre-

  This invention is limited only to deposited microorganisms and viruses, since what is deposited is a mere illustration of one aspect of the invention and all functionally equivalent microorganisms or viruses 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 recombinant viruses, to their chimeric genomes and to proteins or peptides and to

  vaccine formulations, described and shown.

  It is also self-evident that all the dimensions given for nucleotide base pairs are approximate information provided primarily for descriptive purposes.

253 7720

Claims (37)

  1. A recombinant virus according to any one of the   1, 2, 4 and 6 of the main patent, the genome of which comprises a nucleotide sequence coding for an epitope of adenopathy-associated virus (LAV) / leukemia leukemia virus.   human T cells or lymphocytes (HTLV-III). or a   antigenic sequence of this nucleotide sequence which regulates   the expression of the gene so that a peptide, or a protease   related to the epitope of LAV / HTLV III be expressed   in a host infected with the virus, this virus being characteristic of   in that the nucleotide sequence coding for the epitope of LAV / HTLV III comprises the LAV / HTLV III gag gene sequence or any part of this gene coding for a peptide   antigenic or antigenic protein.   2. The recombinant virus according to claim 1, characterized   characterized in that the nucleotide sequence of gag gene comprises the nucleotide sequence substantially as shown in Figure 14 from nucleotide n 340 to 1835, or any portion of that sequence encoding an antigenic peptide or protein antigenic.   3. The recombinant virus according to claim 1, characterized   in that the peptide or protein expressed in the host   infected is related to an epitope of LAV / HTLV III core protein or any antigenic part of this epitode.   4. The recombinant virus according to claim 3, characterized   in that the epitode comprises a peptide or a protein   having an amino acid sequence substantially as   FIG. 14 from the residue of amino acids n 1 to   500, or any antigenic part of this sequence.   5. The recombinant virus according to claim 1, characterized   rised in that it is the recombinant Baculovirus Ac-gagl, as deposited at the American Type Culture Collection (ATCC) and which   received the accession number VR-2147 or a mutant, a recombinant   nant or a derivative of this genetically engineered virus.   6. Sequence v-env 7 of recombinant vaccinia virus se-   claim 7 of the main patent, such that this virus is deposited at the American Type Culture Collection (ATCC) and 101 ', 2587720,   11 was assigned the accession number R 2148, or a mutant, recombinant   or derived from this genetically engineered virus.   7. Sequence v-env5NY of recombinant vaccinia virus   such as this virus was deposited at the American Type Culture Col-   ATCC) and received the accession number VR 2149, or mutant, recombinant or derived from this genetically engineered virus. 8. Ac-env5 sequence of recombinant baculovirus, such as this virus is deposited at the American Type Culture Collection (ATCC) and has received the accession number VR 2151, or a mutant,   recombinant or derived from this genetically engineered virus.   9. Sequence v-gaglNY of recombinant vaccinia virus,   such as this virus was deposited at the American Type Culture Col-   (ATCC), and has received accession number VR 2150, or a mutant, a combination thereof or a derivative thereof. by genetic engineering.   10. Peptide or protein according to claims 18   and 22 of the main patent, this peptide or protein having been purified from one or more cells   part of an animal cell line containing a sequence   this of nucleotides encoding the peptide or protein that   is under the control of a second nucleotide sequence   guiding the expression of genes. so that the peptide or protein is expressed by the cell (s) so cultured, peptide or protein characterized in that it was produced by animal cells infected with v-env5 of recombinant vaccinia virus, such as this virus has been deposited at the American Type Culture Collection (ATCC) and has received the accession number VR 2113, or a mutant, recombinant or derived   of this genetically engineered virus. -   Peptide or protein according to claim 18,   and 22 of the main patent, characterized in that he or she   was produced by cells infected with v-env2 of   recombinant vaccinia rus, such as this virus was deposited at   the American Type Culture Culture (ATCC) and received the nu-   R 2114 input mole or a mutant, recombinant or   this virus obtained by genetic engineering.   12. Peptide or protein according to claims 18   and 22 of the main patent characterized in that he or she was produced by v-env7 infected animal cells of recombinant vaccinia virus, such that this virus was deposited at the American Type Culture Collection (ATCC) and received the accession number VR2148, or a mutant, a recombinant or a derivative of this genetically engineered virus.   13. Peptide or protein according to claims 18   and 22 of the main patent1characterized in that he or she   has been produced by animal cells infected with   env5NY of recombinant vaccinia virus, such as this virus has been deposited at the American Type Culture Collection (ATCC) and there   received the accession number VR 2149, or a mutant, a recombinant   nant or a derivative of this genetically engineered virus.   i4. ept - de - u pr - ot - ine according to claims 18   and 22 of the main patent, characterized in that the   The cultivated lule is or comprises an insect cell line.   and that the peptide or protein has been produced   by insect cells infected with Baculo- Ac-env5   recombinant virus, such as this baculovirus has been deposited at   the American Type Culture Collection (ATCC) and received the   Entry MI VR 2151, or a mutant, recombinant or derivative   of this genetically engineered virus.   Peptide or protein according to claim 15 of the main patent, characterized in that its epitope comprises   an epitope of LAV / HTLV III core protein.   16. Peptide or protein according to claim 15,   characterized in that it comprises a sequence of amino acids   taking the amino acid sequence substantially as   shown in Figure 14 from an amino acid residue number 1 to 500 or any antigenic portion thereof sequence.   Peptide or protein according to claims 18   and 22 of the main patent, characterized) in that he or she   has been produced by animal cells infected with   recombinant vaccinia virus gaglNY, such as this virus was deposited at the American Type Culture Collection (ATCC) and received there accession number VR 2150, or a mutant, a recombinant   or a derivative of this genetically engineered virus.   18. Peptide or protein according to claims 18 and   22 of the main patent, characterized in that the cell cul-   tive is or includes an insect cell line and   whether the peptide or protein has been produced by   lules of insects infected with Ac-gagl baculovirus recom-   such as baculovirus is deposited at the American Type Culture Collection (ATCC) and received the VR accession number   2147, or a mutant, a recombinant or a derivative of this baculo-   virus obtained by genetic engineering.   19. Peptide or protein according to one of the claims   or 16, characterized in that the peptide or protein   was obtained by chemical synthesis.   20. Formulation of live virus vaccine, characterized in that it comprises an infectious dose of the sequence   vaccinia virus v-env7 gene according to claim 6.   21. Formulation of live virus vaccine, characterized in that it comprises an infectious dose of v-env5NY of   vaccinia virus according to claim 7.   22. Formulation of multivalent live virus vaccine, characterized in that it comprises a recombinant virus according to   any of claims 2 to 5 of the main patent   and a recombinant virus according to any of the claims   1 to 11 of this Certificate of Addition, the two viruses recom-   binants being infectious without causing disease to the host to vaccinate.   23. The multivalent vaccine according to claim 22, wherein   characterized in that each recombinant virus comprises a virus with envelope.   24. Multivalent live virus vaccine according to claim   cation 23, characterized in that each envelope virus is or comprises a vaccinia virus.   25. Multivalent vaccine formulation, characterized in that it comprises an infectious dose of: (a) v-env5 of the vaccinia virus according to claim 8, v-env2 according to claim 9, of the main patent, venv7 according to claim 6 of this Certificate of Addition or   v-env5NY according to claim 7 of this Certificate of Ad-   and, (b) v-gaglNY of the vaccinia virus according to the claim   9 of this Certificate of Addition.   26. Inactive virus vaccine formulation, characterized 104 2587720   in that it comprises an effective dose of the v-env7 sequence of the vaccinia virus according to claim 6, in a non   infectious. in admixture with a pharmaceutical excipient.   27. Formulation of inactivated vaccine, characterized by   in that it comprises an effective dose of the v-env5NY sequence of vaccinia virus according to claim 7, at least one   non-infectious condition, in admixture with a pharmaceuti excipient.   28. Formulation of inactivated virus multivalent vaccine, characterized in that it comprises an effective dose of: (a) the recombinant virus according to any one of   claims 2 to 5 of the main patent, which expresses the epi-   tope related to LAV / HTLV III envelope; and (b) the recombinant virus according to any one of   Claims 1 to 4 of this Certificate of Addition, which   expresses the epitope, related to the LAV / HTLV III nucleus, each recombinant virus being in a non-infectious state,   mixture with a pharmaceutical excipient.   29. Formulation of inactivated virus multivalent vaccine, characterized in that it comprises an effective dose of: (a) v-env5 of the vaccinia virus of claim 8 of the main patent, v-env2 of claim 9 of the main patent , v-env7 of claim 6 of this Certificate of addition, or v-env5NY of claim 7 of this Certificate of addition, and (b) v-gaglNY of vaccinia virus of the claim   9 of this Certificate of Addition.   30. Subunit vaccine formulation, characterized in that the immunogen comprises an effective dose of the peptide   or the protein according to any one of the claims   to 22 of the main patent or 10 È 14 of this Certificate   of addition, in admixture with a pharmaceutical excipient.   31. A multivalent subunit vaccine formulation, characterized by comprising an effective dose of: (a) a first peptide or a first protein according to claim 16 or 17 of the main patent; and (b) a second peptide or a second protein according to   any one of claims 19 to 22 of the main patent   pal or 14 to 18 of this Certificate of Mixed Addition with a pharmaceutical excipient.   32. DNA vector (deoxyrobonucleic acid) recombinant   nant, characterized in that it is or comprises pv-env7.   33. Recombinant DNA vector, characterized in that it is or comprises pv-gag1. 34. Recombinant DNA vector, characterized in that is or understands pAc-gagl.   35. Recombinant DNA vector, characterized in that is or understands pAc-env5.   36. Unicellular organism, characterized in that it   holds the recombinant DNA vector of any of the claims 32 to 35.   37. Bacteria containing the recombinant DNA vector se-   any one of claims 32 to 35   38. A bacterium according to claims 37 and 32, characterized   rised in that it is or comprises Escherichia coli deposited at NRRL and which has been assigned the accession number B-18110, or a mutant,   a recombinant or genetically engineered derivative.   39. A bacterium according to claims 37 and 33, which is   in that it is or comprises Escherichia coli deposited at NRRL and which has been assigned the accession number B-18111, or a mutant, a recombinant or a derivative of this bacterium obtained by genetic engineering.   40. A bacterium according to claims 37 and 34, characterized   A recombinant Escherichia coli deposited at NRRL and which has been assigned the accession number B-18105, or a mutant, a recombinant or a derivative of this genetically engineered bacterium.   41. A bacterium according to claims 37 and 35, characterized   in which it is or comprises Escherichia coli deposited at NRRL and which has been assigned the accession number B-18109, or is a mutant, a recombinant or a derivative of this bacterium obtained by genetic engineering.