FR2593519A1 - Vaccine against the acquired immunodeficiency syndrome and intermediates for preparing this vaccine - Google Patents

Abstract

The invention relates to recombinant viruses which direct the expression of peptides or proteins similar to epitopes of the adenopathy virus LAV and human T lymphocyte leukaemia virus HTLV III, which is the causative agent of the acquired immunodeficiency syndrome AIDS. These viruses can be formulated in the form of vaccines containing live or inactivated viruses or in polyvalent vaccines for protecting against infection caused by LAV/HTLV III. Peptides and proteins similar to epitopes of this virus are also described. They can be prepared using recombinant DNA techniques or by chemical synthesis and used as immunogens in subunit vaccines or as antigens in diagnostic immunological assays.

Description

The present invention relates to the preparation of
vaccines against acquired immunodeficiency syndrome (AIDS),
as well as the vaccines obtained and also the various
intermediaries used for this purpose.

Thus, the present invention relates to viruses
which express peptides and proteins related to
epitopes of the lymphadenopathy / adenopathy-associated virus
Associated Virus (LAV) 7 / Leukemia Cell Virus
T Human / Human T-Cell Leukemia Virus (HTLV-III) /, the agent
etiological syndrome of lymphadenopathy / Syn lymphadenopathy
drome (LAS) 7 and acquired immunodeficiency syndrome (AIDS); / Acquired Immune Deficiency Syndrome (AIDS) 7.Viruses
The present invention can serve as immunogens in
virus-based vaccine formulations for the syndrome
lymphadenopathy (LAS) or AIDS or in
multivalent vaccines.In fact, the infectious viruses of the
present invention, which can multiply in a host
without causing disease, can be used in formulas
Vaccines based on live viruses that provide a
prolonged immunogenic stimulation and can give birth
to a high immunity.

The present invention also relates to peptides
and proteins related to epitopes of LAVIHTLV III, for
to serve as immunohullenes in vaccine formulations
subunits for LAS or AIDS, or in formulations
of multivalent vaccines, or as antigens in assays
immunological tests for the diagnosis of LAS or AIDS. These
peptides and proteins can be produced using
techniques using recombinant DNA in
any host-vector system, or they can
be synthesized by chemical methods.So, the inven
The aim is also to build new sequences of
DNA and vectors comprising plasmid DNA, and
Viral DNA 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 an appropriate 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 embodiment of the present invention, a recombinant vaccinia virus has been used to produce LAV / HTLVIII related proteins.

For this, DNA sequences encoding the LAV / HTLV III glycoproteins were inserted into a vaccinia vector capable of directing the expression of the LAV / HTLV III glycoprotein gene in a suitable host. The LAV / HTLV III proteins produced by the recombinant vaccinia virus have been found to be antigenic and immunogenic.

 These recombinant vaccinia viruses can themselves be used in virus vaccine formulations.

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

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

 AIDS (Acquired 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: 1431) .Two clinical presentations of the disease are recognized: (a) a prodrome phase, known as lymphadenopathy / Syndrome syndrome ( LAS) 7, characterized by chronic lymphadenopathy, leukopenia and a quantitative decrease of peripheral blood helper cells (OKT4 cells), leading to a reversal of the normal ratio between helper lymphocytes and suppressor T 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 Kaposi's lymphoma and sarcoma.

Epidemiological data and information on the types of patients who 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 to lymphocytes.
T auxiliaries. These groups are
(a) RC Gallo and his staff at the National
Institute of Health were able to isolate a cytopathic retrovirus (HTLV III) on patients with AIDS and pre
AIDS (RC 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 AIDS patients.

(b) L. Montagnier and his collaborators isolated at the Institut Pasteur a lymphotropic retrovirus (LAV) 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 in serum from AIDS patients (Kalyansraman, VS, et al., 1984, Science 225: 321). In addition, they were able to isolate LAV from lymphocytes of a patient who had developed
AIDS after receiving blood from a donor with
AIDS (PM Feorino et al., 1984, Science 225: 69).

 (c) Levy J. et al. isolated infectious retroviruses (referred to as AIDS-associated retroviruses (ARVs)) from peripheral mononuclear cells of patients with AIDS (JA 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 will be collectively referred to herein as LAV / HTLV III.

The general structure of retroviruses is that of a nucleus of ribonucleoproteins surrounded by a lipid-containing envelope that the virus acquires during cellular budding. The virally encoded glycoproteins are buried within the envelope and protrude outwardly. They determine the viruses known to the host and react with specific receptors on the surface of sensitive cells. Neutralizing antibodies are thought to bind to envelope glycoproteins and block their interaction with receptors on the surface of cells (pages 534-535 in "The Molecular
Biology of Tumor Viruses ", published by John Tooze, 1973, Cold Spring Harbor
Laboratory; pages 226-277 and 236-237 in "RNA Tumor Viruses" (RNA Tumor Virus), published by
R. Weiss, N. Teich, H. Varmus and J. Coffin, 1982, Cold
Spring Harbor Laboratory). In the specific case of LAV /
HTLV III, there is evidence that antigen
T4, present in a subgroup of T cells, is the receptor or component of the virus receptor (AG

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

 The RNA genome of LAVIHTLV 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 antigens specific to the group of viruses. The pol gene codes for virion-associated reverse transcriptase. The env gene codes for viral glycoproteins. 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. These include vaccines that trigger an active immune response, treatment with chemotherapy agents, and interferon therapy.

 Traditional ways of preparing vaccines include the use of inactivated or attenuated viruses.

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

 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 are then chosen which have lost virulence but are still capable of eliciting an immune response. Attenuated viruses are generally good immunogens because they replicate in the host cell and trigger long-lasting immunity.

However, there are several problems with the use of live vaccines, the most troublesome of which is insufficient attenuation.

Instead of the above methods, so-called subunit vaccines, or subunits of vaccines, may be used. This involves immunization obtained only with the help of the proteins containing the immunological material concerned. For many enveloped viruses, the virally encoded glycoprotein contains epitopes capable of triggering the production of neutralizing antibodies; they include the glycoproteins of the La Crosse virus (F. Gonzalez
Scarano, RE Shope, CE Calisher and N. Nathanson, 1982,
Virology 120: 42.), The neonatal diarrhea virus of the calf (S. Matsuno and S. Inouye, 1983, Infection and Immunity 39: 155), the equine encephalomyelitis case of Venezuela (JH

Mathews and JT Roehrig, 1982, J. Imm. 129: 2763), the Punta Toro virus (JM Dalrymple, CJ Peters, JF Smith and
MK Gentry, 1981 in "Replication of Negative Strand
Viruses "(Replication of Strand negative virus), DHL

Bishop and R.W. Compans, page 167, Elsevier, New York), mouse leukemia virus (R. A. Steeves, M. Strand and J.T.

August, 1974, J. Virol. 14: 187) and "Mouse Mammary Tumor Virus" (RJ Massey and G. Schochetman, 1981, Virology 115: 20). An advantage of subunit vaccines is that the unattractive viral material is excluded
Vaccines are often given in combination with various adjuvants. Adjuvants help achieve a more sustainable and higher level of immunity by using smaller amounts of antigens at lower doses than if the immunogen was administered alone. The mechanism of action of adjuvants is complex and not fully understood. However, it may involve stimulation of phagocytosis and other activities of the reticuloendothelial system as well as delayed release and degradation of the antigen. Examples of adjuvants include Freund's adjuvant (complete or incomplete) , adjuvant 65 (containing peanut oil, mannide monooleate and aluminum monostearate), polyol "Pluronic L-121", "Avridine", and mineral gels such as sodium hydroxide. aluminum, aluminum phosphate or
Moon. Freund's adjuvant is no longer used in vaccine formulations for humans because it contains non-metabolizable mineral oil and is a potential carcinogen.

 The use of recombinant DNA technology for the production of subunit vaccines involves molecular cloning and expression in an appropriate vector of the viral genetic information encoding the proteins capable of eliciting 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 is not likely to become infected.

Recently, a new approach has been described which is potentially useful for the production of subunit vaccines (Mackett, GL Smith and B. Moss, 1982).
Proc. Nat'l. Acad. Sci. 79, 7415-7419; Mr. Mackett, GL

Smith and B. Moss, 1984, J. Virol. 49, 857-864; D. Panicali and E. Paoletti, 1982, Proc. Nat'l. Acad. Sci. 79, 4927-4931).

This approach involves the use of vaccinia virus as a vector for expressing foreign genes inserted into its genome. When introduced into animal hosts, the recombinant vaccinia virus expresses the inserted foreign gene and thereby triggers an immune response of the host to such genes. Since the recombinant live vaccinia virus can serve as a vaccine, such an approach combines the advantage of subunit vaccines and live vaccines.

The vaccinia virus contains a double-stranded linear DNA (deoxyribonucleic acid) genome with about 167 kilobase pairs and replicates within the cytoplasm of infected cells. These viruses contain a complete system of transcription enzymes (including styling, methylation and polyadenylation enzymes) within the nucleus of the virus, which are necessary for the infectivity of the virus. The vaccinia virus transcriptional regulatory sequences (promoters) allow initiation of transcription by the vaccinia RNA polymerase, but not by the RNA polymerase of the host cells.

Expression of foreign DNA in recombinant vaccinia viruses requires ligation of vaccinia promoters to DNA sequences encoding foreign gene proteins. Plasmid vectors, also called insertion vectors, have been constructed to insert chemical genes into vaccinia viruses. One type of insertion vector is composed of: (a) a vaccinia virus promoter comprising the site transcription initiation; (b) several unique restriction endonuclease cloning sites, located downstream of the transcription start site for the insertion of foreign DNA fragments (c) of non-essential vaccinia virus DNA (such as the gene
TK) surrounding the promoter and cloning sites that direct 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 antibiotic resistance 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, 857864).

 Recombinant vaccinia viruses are produced by transfection of recombinant bacterial insertion plasmids, containing the foreign gene of cells previously infected with vaccinia virus. Homologous recombination occurs within the 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 can be constructed to have about 35 kilobases of foreign DNA.

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

Viruses that direct the expression of peptides or proteins related to LAV epitopes / will be described
HTLV III. These viruses can be formulated as virus vaccines to protect humans against LAV / HTLV III infection. In a particular embodiment of the present invention, live virus vaccine formulations can be prepared using infectious viruses that express LAV / HTLV III related epitopes in an infected host, but do not cause such an infection. disease in this host.

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

In a specific embodiment of the present invention, the gene coding for LAV / HTLV III envelope glycoproteins (env gene of LAV / HTLV III) is inserted into a plasmid such that a virus promoter of Prior vaccinia is placed 5 'to the initiating methionine sequence (ATG) of the env gene LAV / HTLV III, resulting in a chimeric gene which is in turn flanked or surrounded by thymidine kinase (TK) sequences. of
Vaccinia DNA. This plasmid is then transfected (introduced) into cells that have been previously infected with wild-type vaccinia virus, which allows the LAV / HTLV III env chimeric genes, surrounded by the TK sequences, to recombine in the cell. TK gene of vaccinia virus.

The cells are allowed to lyse and the resulting virus is cultured on TK- cell plates. The recombinant virus was selected based on its ability to bind to cultures of these plaque cells in the presence of 5-bromo-deoxyuridine as well as by DNA-DNA hybridization on a radiolabeled LAV / HTLV III envelope probe.

Hybrid-positive plaques were purified, grown, and the resulting recombinant virus assayed for production ability of LAV / HTLV III envelope glycoproteins. Virus stocks, or virus reservoirs, are used to produce these proteins to infect the test animals. Serum obtained from these animals can be assayed for antibodies against LAV / HTLV III glycoprotein, to determine the ability of this serum to prevent viremia caused by
LAV / HTLV III, and finally to detect the ability to confer protection against AIDS.

 The accompanying figures will now be briefly described.

 Figure 1 shows the structure of the integrated proviral genome of LAV / HTLV III. Hatched areas indicate regions of open reading frames. The regions coding for the group-specific antigen, the reverse transcriptase and the envelope proteins are designated respectively by gag (antigen-specific group), pol and env.

The overlapping open-reading frames are represented by cross-hatched areas. Numbers refer to numbers of base pairs downstream of the styling site ("cap" site), or start viral transcription.

Restriction sites are marked as follows: Bg,
BglII; Ec, EcoRI; Hn, HindIII; kp, kpnI; Ss, SstI.

Figure 2 shows the nucleotide sequence of the LAV specific region (EcoRI to SstI) present in the plasmid pRS-3 DNA; the entire envelope gene of
LAV (nucleotide 5766 to 8349) is contained in the insertion
LAV. The restriction sites used in the construction of pv-env, 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 the plasmid construct containing a portion of the sequence coding for LAV envelope proteins, a sequence inserted downstream of a vaccinia virus promoter. The coding sequence for the LAV 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 envelope is shown at the bottom of the figure. The underlined sequences indicate the presumed priming codons and the reading frame for the chimeric gene. Note that the third and fourth amino acids of the translated sequence (Pro-Val) of recombinant pv-env2 correspond to amino acids number 43 and 44 of the LAV envelope coding sequence.

Figure 4 is a schematic representation of the plasmid construct containing the entire LAV envelope protein coding sequence inserted downstream of a vaccinia virus promoter. The vaccinia virus promoter is represented by the hatched or shaded bar. Plasmid pv-env5 containing the entire LAV 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-env1 which contains the amino terminus of LAV envelope gene coding and pv-env2 which contains the carboxyl end of coding of the LAV envelope gene. These two portions are grouped together at the StuI site as shown,
Figure 5 shows the construction and selection of recombinant vaccinia virus. The solid bars represent the vaccinia virus thymidine kinase (TK) gene. 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 vaccinia 7.5K promoter (hatched bar) and by the coding region of the plasmid. LAU envelope (open bar). After infection of the cells with vaccinia, the recombinant plasmid containing the gene
TK interrupted by the LAV envelope gene is introduced into the infected cells. The recombinations that occur in the TK sequencers surrounding the chemical gene introduce the LAV envelope gene sequence into the vaccinia virus genome. The resulting recombinant virus, containing the LAU envelope gene, is TK
Figure 6A shows Western immunocoagulation analysis of proteins produced by vaccinia env-LAV recombinants. Proteins from the following sources were resolved on a gradient polyacrylamide gel of 7 to 15% SDS and electrotransferred onto nitrocellulose paper: virion proteins.
LAV (LAV); cells infected with wild-type vaccinia virus ("WTvv") ("wild-type vaccinia virus"); uninfected cells ("mock"); cells infected with recombinant viruses v-env2 (v-env2) or v-env5 (v-env5). Immunoreactive proteins with respect to the serum of a patient with AIDS were decelerated by the action of a peroxidase conjugated to anti-human IgG antibodies. The LAV / env genes produced are indicated by gp150, gp101 and gp41. Molecular weight standards are expressed in kilodaltons (kd).

 Figure 6B depicts a Western immunocoagulation assay of proteins produced by LAV-env vaccinia recombinants in two cell types. Proteins from BSC-40 or HeLa cells were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto nitracellulose paper and then reacted with pooled serum from positive serum recipients. for LAV / HTLV-III lanes 1 and 5 represent uninfected "mock" cells; 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 125 I-labeled protein A. The LAV-env gene products are indicated by gap 150, gp10, and gp41.

Figure 7A 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 AIDS patient serum (I) and the immune complexes were precipitated by protein A
Staphylococcus aureus. The immunoprecipitated proteins were resolved by electrophoresis on a 15% SDS polyacrylamide gel and detected by fluorography. Molecular weights are given in kilodaltons (kd).

 Figure 7B shows the results of radioimmunoprecipitation analysis of 3Hglucosamine labeled proteins produced by the vaccinia-LAV recombinants of the invention. Hela cells were either infected with non-infectious cells (mock "non-infection") (lanes 1 and 5) or infected with wild-type vaccinia viruses (lanes 2 and 6), v-env5 (lanes 3 and 5). 7) or v-env2 (lanes 4 and 8) and labeled with 3H-glucosamine.

Cell lysates were immunoprecipitated with normal human serum (lanes 1-4) or with pooled serum from LAV positive serum individuals /
HTVL III (lanes 5-8) and Protein A. The immunoprecipitated proteins were resolved by SDS-polyacrylamide gel electrophoresis.

Figure 7C shows the results of a "pulse-hunting" radioimmunoprecipitation analysis of LAV envelope proteins produced by LAV vaccinia recombinants. 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 time, the cells were washed, lysed and immunoprecipitated with pooled serum from serum positive individuals for
LAV / HTLV III and resolved by SDS-PAGE (polyacrylamide gel electrophoresis): O hour (lanes 1, 7, 13) half an hour (lanes 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).

Figure 7D shows the results of a radioimmunoprecipitation analysis of LAV-related proteins found in cells and in
media from virus-infected cells
Recombinant vaccinia-LAV of the invention. Cells
HeLa were either infected with uninfected viruses
(ie non-infected, track 1 "mock"), either infected
by wild-type vaccinia virus (lane 2) v-env5
(lane 3) or v-env2 (lane 4) and labeled with 35 S-methionine.

The cells were separated from the medium and lysed. Lysates
of cells (pellet) and middle (supe) (laughing) were
each immunoprecipitated with pooled serum from
of people with serum positive for LAV / HTLV III. The
Immunoprecipitated proteins were resolved by electropho-
polyacrylamide gel (SDS-PAGE).

Figure 8 shows an immunocoa analysis
Western regulation of serum samples from
mice immunized with recombinant vaccinia viruses
LAV. The mice had been immunized with
recombinant vaccinia v-env5 or v-env2. After 8 weeks,
the serum samples were reacted with proteins
virion ines LAV that had been solved by electropho
on SDS-PAGE (polyacrylamide gel) and electrotrans
made on nitrocellulose paper. We used a
goat anti-mouse immunoglobulin conjugated to alkaline phosphatase to detect LAV proteins that have
have been recognized by the sera of mice.
represent serum samples from 5 individual mice
duels that were inoculated v-env5, and tracks 9
k represent serum samples from 5
individual mice inoculated with v-env2. The
grouped sera from persons with positive result
for LAV / HTLV III (AIDS) and non-immunized mice C57B16J
(NMS) served as positive and negative controls, respectively
is lying. The positions of the envelope glycoproteins LAV gp150,
gel 10 and gp43 are indicated.

Figure 9 shows an immunocoa analysis
Western regulation of serum samples from
macaques immunized with recombinant vaccinia virus LAV.

 Four macaque monkeys (numbers 168, 175, 180, 182) were inoculated with 2x108 vaccinia herpes simplex recombinant plaque formation units gD (v-HSgD1). Serum samples were collected prior to inoculation (lanes p) and after 3, 4 and 6 weeks after inoculation (lanes 3, 4 and 6, respectively). Serum aliquots were diluted 50-fold and reacted with LAV virion protein which was resolved by polyacrylamide gel electrophoresis (SDS-PAGE) and immobilized by electrotransfer on nitrocellulose filters. The LAV proteins recognized by these sera were detected by goat anti-human immunoglobulin conjugated to alkaline phosphatase. Grouped sera from LAV / HTLV III positive persons (AIDS) served as a positive control.

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

The present invention aims to obtain viruses that produce peptides and proteins related to LAV / HTLV III epitopes. The invention also relates to peptides and proteins related to LAV / HTLV epitopes
III, which can be produced using methods using recombinant DNA or by chemical synthesis. The viruses or peptides and proteins of the present invention can serve as immunogens in various vaccine formulations, which include multivalent vaccine formulations to protect against infection with LAV / HTLV III, which is the causative agent of LAS (Syndrome). lymphadenopathy) and AIDS
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. sequences of
LAV / HTLV III in suitable 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 and serve as an immunogen in subunit vaccine formulations.

Alternatively, the amino acid sequences of these peptides and proteins can be deduced from LAV / HTLV III nucleotide sequences contained in recombinants that express LAV / HTLV III-related immunogenic peptides and proteins. These peptides and proteins can then be chemically synthesized and used in synthetic subunit vaccine formulations.

 When the expression vector is a virus, the virus itself can be formulated as a vaccine. Infectious recombinant viruses, which do not trigger disease in the host, can be used in live virus vaccine preparations to confer significant immunity. Alternatively, vaccines containing inactivated viruses can be prepared when "killed" viruses are used. In addition, multivalent vaccines containing LAV / HTLV III epitopes as well as other disease-causing agents can be prepared.

 For a clear description, the method 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 a specific embodiment of the invention, the construction of recombinant vaccinia virus containing the LAV / HTLV III envelope gene which directs the expression of proteins immunologically bound to LAV envelope proteins is described. HTLV III in tissue culture cells infected with recombinant viruses.

However, the compositions and methods described herein are not limited to the construction of recombinant vaccinia viruses expressing LAV / HTVL III-related proteins, and can be used to reconstruct recombinants in any which expression vector system for the production of antigen-related polypeptides of any etiologic agent of AIDS.

For the sake of clarity, the entire process will be studied in terms of LAV / HTLV envelope genes.
III. However, the same technique 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.

ISOLATION OF GENES, OR GENE FRAGMENTS, ENCODING
VIRAL PROTEINS LAV / HTLV III
The isolation of the LAV / HTLV III envelope gene first involves the isolation of DNA fragments containing the envelope gene sequences. As previously explained, LAV / HTLV III has an RNA genome and, therefore, the corresponding DNA that encodes the gene can be obtained.
LAV / HTLV III (a) by cloning the purified virion RNA isolated from LAV / HTLV III by cDNA, or (b) cleaving polyA-containing RNA from cDNA infected cells by cDNA. LAV / HTLV III, or (c) by cloning purified genome DNA from LAV / HTLV infected cells
III. Hereinafter, the DNA encoding the LAV genes will be called
HTLV III of LAV / HTVL III DNA.

In order to generate DNA fragments of LAV / HTVL
III, the AV / HTLV III DNA can be cleaved at specific sites using various restriction enzymes.

Alternatively, ase DNA can be used in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, for example by sonication.

The linear DNA fragments can then be separated according to their dimensions by conventional techniques, which includes, but is not limited to, agar and polyacrylamide gel electrophoresis and column chromatography.

 Any restriction enzyme or combination of restriction enzymes may be used to generate one or more LAV / HTLV III DNA fragments containing the envelope sequence, provided that the enzymes do not destroy the immune power of the envelope gene protein product. For example, the antigenic site of a protein may be from about 7 to about 14 amino acids. Thus, a protein of the size of the envelope precursor peptide (about 97,000 daltons) may have many separate antigenic sites, perhaps thousands considering the overlapping sequences, secondary and tertiary structural considerations, and potential Thus, many polypeptide gene sequences of envelopes may encode an antigenic site. Therefore, many combinations of restriction enzymes can be used to generate DNA fragments which, when inserted into an appropriate vector, are capable of directing the production of envelope-specific amino acid sequences comprising different antigenic determinants. .

Once the DNA fragments have been generated, an identification of the specific DNA fragment containing the envelope gene can be carried out in a number of ways.
LAV / HTLV III. First, it is possible to sequence the DNA fragments corresponding to the entire genome of LAV / HTLV III and then identify the fragments containing the envelope protein gene sequence based on a comparison of the sequence of predicted amino acids and the amino acid sequence of the envelope protein.

Secondly, once the entire genome sequence has been determined, the large open reading frames can be ordered from 5 'to 3'. Since the genome organization of all the retroviruses examined so far is 5'-gagpol -env-3 ', the large open reading frame closest to the 3' end is very likely to encode the envelope gene. Alternatively, the fragment containing the envelope protein gene can be identified by selection of mRNA. In this procedure, the LAV / HTLV III DNA fragments are used to isolate, by hybridization, the complementary mRNAs. Immunoprecipitation analysis of the in vitro translation products of the isolated mRNAs identifies the mRNA and thus the complementary LAV / HTLV III DNA fragments that contain the envelope protein sequences. Finally, protein-specific mRNAs can be selected. polysome adsorption coat isolated from LAV / HTLV III infected cells on immobilized antibodies directed against the envelope protein.

Radiolabeled envelope DNA, cDNA (complementary DNA) can be synthesized using the selected mRNA (from the adsorbed polysomes) as a template. Radiolabeled mRNA or radiolabeled cDNA can then serve as a probe for identifying LAV / HTLV III DNA fragments containing envelope protein gene sequences. Instead of isolating the envelope gene, it is possible, without this list being limiting, to chemically synthesize the gene sequence itself (provided that the sequence is known) or to transform cDNA into mRNA that encodes the gene. envelope gene.

Once identified and isolated, the DNA fragment
LAV / HTLV III containing the sequences of interest can be first inserted into a cloning vector, such as a plasmid cloning vector, which serves to transform appropriate host cells to cause replication of the DNA to generate many copies of the interesting LAV / HTLV III sequences. This can be achieved by ligating the LAV / HTLV DNA fragment.
III in a cloning vector having complementary cohesive terminations. However, if the complementary restriction sites used to fragment the DNA of LAV /
HTLV III are not present in the cloning vector, the ends of the DNA molecules are likely to be modified. Such modifications include the production of blunt ends by digestion of the termini.
Single-stranded DNA or single-stranded terminations 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 methods, the cleaved vector and the L-AV / HTLV III DNA fragment can be terminated by homopolymer termination.

Transformation of host cells by recombinant DNA molecules that incorporate the isolated gene, by
CDNA or a synthetically obtained DNA sequence makes it possible to generate multiple copies of the gene. Thus, the gene can be obtained in large amounts by growth of transformants, isolation of recombinant DNA molecules from the transformants and, when necessary, recovery of the inserted gene from the isolated recombinant DNA.

 If the ultimate goal is to insert the gene into virus expression vectors such as vaccinia virus or adenovirus, the recombinant DNA molecule that incorporates the LAV / HTLV III gene can be modified so that the gene is surrounded by virus sequences allowing genetic recombination 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 Wain-Hobson 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 LAU envelope gene, is pRS-3 which consists of a .about.840 base pair EcoRI to SstI fragment of the LAV nucleotide sequence inserted into the EcoRi and SstI site. of pUC18. The specific DNA of LAV contained in pRS-3 is located between the site
EcoRI placed at nucleotide 5289 and the SstI site placed at nucleotide 9129 on the LAV genome (Wain-Hobson et al., 1985, Cell 40: 9); see Figure 2 which shows the nucleotide sequence of LAV contained in pRS-3. However, because of the degeneracy of the nucleotide coding sequences, it is possible to use in the practice of the present invention, for the cloning of the gene of envelope of LAV / HTLV
III, other DNA sequences that encode substantially the same amino acid sequence as that shown in Figure 2. This includes, but is not limited to, nucleotide sequences comprising all or portions of the nucleotide sequence of envelope shown in Figure 2, which are modified by replacing different codons that 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.

INSERTING ENCODING SEQUENCES OF LAV / HTLV PROTEINS
III IN EXPRESSION VECTORS
The nucleotide sequence encoding the LAV / HTLV III coat protein, or part thereof, is inserted into an appropriate expression vector, ie a vector which contains the necessary elements for the expression of the LAV / HTLV III envelope protein. transcription and translation of the coding sequence of inserted proteins. Various host systems can be used to express the protein coding sequence.

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. Depending on the host-vector system used, any one of a number of suitable transcription and translation elements may be used. For example, when cloning into mammalian cell systems, promoters isolated from the genome of mammalian cells (e.g., a mouse metallothionene promoter) or isolated from viruses grown in these cells (e.g. vaccinia virus). It is also possible to use promoters produced by recombinant DNA or by synthetic techniques to ensure the transcription of inserted sequences.

Specific bootstrapping signals are also required for efficient translation of the inserted protein coding sequences. These signals include the boot codon
ATG and the adjacent sequences. When the entire LAV / HTLV III envelope gene, including its own priming codon and adjacent sequences, is inserted into the appropriate expression vectors, additional control signals for translation may not be necessary.

However, when only a portion of the envelope coding sequence is inserted, exogenous translational control signals, which include the ATG translation initiation codon or initiation, must be provided. The initiation or initiation codon must also be in phase with the reading frame of the envelope protein coding sequences to ensure the translation of the entire inserted sequence.

These exogenous translational control signals and translation initiation or initiation codons can have various origins, both natural and synthetic.

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

In the particular embodiment described in detail in the examples of the present invention, the vaccinia virus has been chosen as an expression vector. However, the invention is not limited to the use of vaccinia virus. As previously explained, usable expression vectors include, but are not limited to, the following vectors or their derivatives: viruses infecting humans or animals such as vaccinia virus or adenoviruses; viruses infecting insects such as baculoviruses; yeast vectors; vectors of the bacteriophage type, and vectors of the type
Plasmid DNA and cosmid to name a few.

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

 Another expression system that could be used to express the LAV / HTLV III glycoprotein is a system derived from insects. In such a system, Autagrapha californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes. The virus is believed to be in Spodoptera frugiperda cells. The LAV / HTLV III envelope gene can be cloned into non-essential regions (e.g. the polyhedrin gene) of the virus and this is placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter). Successful insertion of the LAV / HTLV III gene will result in inactivation of the polyhedrin gene and production of an unclosed recombinant virus (i.e., a virus lacking the encoded protein coat for the polyhedrin gene).

These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed.

 In addition, one may select a host cell strain that modulates the expression of the inserted sequences or modify and process the chimeric gene product in the specific manner desired. The expression of certain promoters can be increased in the presence of certain inducers (for example zinc and cadmium ions for metallothionein promoters). Thus, expression of the genetically engineered LAV / HTLV III-env protein can be regulated. This is important if the protein product of the cloned foreign gene is lethal to the host cells. In addition, modifications (e.g., glycosylation) and treatment (e.g., cleavage) of the protein products are important for the functioning of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification. proteins. Appropriate cell lines or host systems may be selected to ensure the correct modification and processing of the expressed foreign protein.

 In the particular embodiment described in detail in the examples of the present invention, the sequences encoding the LAV envelope (LAV env sequence), both in their complete form and in their portions, were ligated onto the promoter. 7.5K vaccinia virus to form chimeric genes in various plasmids. The chimeric genes of these plasmids were surrounded by additional vaccinia virus sequences homologous to the vaccinia virus TK gene. The construction of the chimeric gene involved the use of nucleotides, both natural and synthetic, encoding control signals for transcription and translation of LAV envelope sequences. The chimeric gene was then introduced into the vectors. of vaccinia virus expression by in vivo recombination between the homologous TK region present on the plasmid vector and the vaccinia virus genome. These recombinant viruses, containing the chimeric gene, were used as expression vectors to produce LAV-related proteins.

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 of inserted sequences. In the first approach, it is possible to detect the presence of a foreign gene inserted in an expression vector by DNA-DNA hybridization using probes comprising sequences which are homologous to the inserted foreign gene. In the second approach, it is possible to identify and selecting the recombinant / host vector system based on the presence or absence of certain "marker" gene functions (e.g., thymide kinase activity, antibiotic resistance, transformation phenotype, etc.). ) due to the insertion of foreign genes into the vector. For example, if the LAV / HTLV III gene is inserted into the sequence of the vector marker genes, recombinants containing the inserted LAV / HTLV III sequence can be identified by the absence of the marker gene function. In the third approach, recombinant expression vectors can be identified by searching for and / or assaying the product of the foreign gene expressed by the recombinant. Such research or assays may be based on the physical, immunological or functional properties of the gene product.

 Once a particular molecule of recombinant DNA identified and isolated, one can use several methods to cause propagation, according to whether such a recombinant is or not a self-replicating unit (a replicon).

A self-replicating unit, for example plasmids, viruses, cells, etc., can grow in the appropriate cellular environment and under appropriate growth conditions. Recombinants lacking a self-replicating unit will have to be integrated into a molecule comprising such a unit for the propagation to take place. For example, certain plasmid expression vectors, when introduced into a host cell, must be integrated with the cell chromosome to ensure propagation 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 the particular embodiment of the invention described in detail in the examples, the chimeric gene containing the region encoding the envelope of
LAV in the TK gene of the vaccinia virus genome, thereby transforming the virus into TK, i.e., destroying the ability of the virus to make thymidine kinase.

Such recombinants were chosen on the basis of their ability to grow in media containing 5-bromo-deoxyuridine, which is a nucleoside analogous to that killing for TK cells but not for TK cells. Recombinants have also been identified by DNA-DNA hybridization, using specific probes of the LAV envelope.

Recombinant TK virus was isolated by plaque purification and reserve stocks were prepared from infected tissue culture cells.

IDENTIFICATION AND PURIFICATION OF THE PRODUCT OF THE EXPRESS GENE
Once a recombinant expressing the LAV / HTLV III gene has been identified, the gene product should be analyzed.

This can be achieved by analyzes and assays based on the physical, immunological or functional properties of the product. An immunoassay is particularly important since the ultimate goal is to use gene products or recombinant viruses that express such products in vaccine formulations and / or as antigens in immunoassays for diagnostics.

 Various antisera are available to analyze the immunoreactivity of the product, which includes, but is not limited to, serum from patients with LAS (adenopathy syndrome) or AIDS and polyvalent sera directed against the LAV / HTLV virus. , the viral coat protein and its analogs produced in a bacterial system, or the synthetic peptides containing antigenic determinants of the LAV / HTLV III envelope. The identification of the peptides and proteins described in the present invention is based on two requirements. Firstly, the LAV / HTLV III envelope-related protein must be produced only in cells infected with recombinant virus. Second, the LAV / HTLV III-related protein must be capable of an immune reaction with the serum of AIDS patients or with various antibodies directed against LAV / HTLV III envelope protein analogues and derivatives.

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

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

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

Hunkapiller et al., 1984, Nature 310: 105-111).

 In a particular embodiment 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 sequence essentially as shown in Figure 2, which includes altered sequences in which functionally equivalent amino acid residues replace residues within the sequence, which creates a silent variation. For example, one or more amino acid residues in the sequence may be replaced by another amino acid of similar polarity, which acts as a functional equivalent, giving a silent alteration. the class to which the amino acid belongs, elements to replace an amino acid within the sequence. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Neutral polar amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged amino acids (acids) include aspartic acid and glutamic acid.

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

The immune response of the test subjects can be analyzed by three approaches: (a) the reactivity of the resulting immune serum with respect to authentic viral antigens of LAV /
HTLV III, according to assays or tests carried out by known techniques, for example bound enzyme immunosorption assay (ELISA), immunocoagulants, radioimmunoprecipitations, etc., (b) the ability of the immune serum to be neutralized in vitro "the infectivity of LAV / HTLV III (M.

Robert-Guroff, 1985, Nature 316: 72-74), and (c) protection against LAV / HTLV III infection and / or attenuation of infectious symptoms in immunized animals (D.P.

Francis, 1984, Lancet 2: 1276-1277; DC 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 related to an epitope of LAV / HTLV III or is a recombinant virus that expresses such an immunogen that protects against LAV virus infections. / HTLV III for the prevention of lymphadenopathy (LAS) or AIDS. In addition, multivalent vaccine formulations can be prepared in which the LAV / HTLV III gene product or a recombinant virus expressing the chimeric gene product, in combination with other immunogens, is used for the prevention of lymphadenopathy (LAS) or AIDS and other diseases. Examples of various formulations are presented below.

VIRUS VACCINE FORMULATIONS
A live recombinant virus vaccine or an inactivated recombinant virus vaccine can be formulated. The choice depends on the nature of the recombinant virus that is used to express LAV / HTLV III related epitopes. When the recombinant virus is infectious to the host to be immunized but does not cause disease, a live vaccine is preferable because a multiplication in the host leads to prolonged stimulation of a similar genus and amplitude. which occurs in natural subclinical infections and, therefore, this confers a high immunity of long duration.When introduced into a host animal, the infectious recombinant virus can express the proteins related to the LAV / STLV III envelope from of its chimeric gene and thereby stimulate an immune response against LAV / HTLV III antigens. When such an immune response provides protection against further attack by LAV / HTLV III, the live recombinant virus can itself serve as a preventative 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.

 Live multivalent vaccines can be prepared from a single or a small number of infectious recombinant viruses that express epitopes of organisms causing other diseases in addition to LAV / HTLV III epitopes. For example, vaccinia virus (which can receive about 35 kilobase of foreign DNA) can be genetically engineered to contain coding sequences of other epitopes in addition to those encoding LAV / HTLV III; such a recombinant virus itself can serve as an immunogen in a multivalent vaccine.

Alternatively, a mixture of vaccinia virus or other viruses capable of directing the expression of a different gene encoding different epitopes of LAV / HTLV III and / or d) can be formulated in a multivalent vaccine. other organisms capable of causing disease.

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

A mixture of inactivated viruses that expresses different epitopes can be used to formulate "multivalent" vaccines.

In some cases, this may be preferable to live vaccine formulations, due to potential difficulties due to mutual interference of live viruses administered together. In either case, the inactivated recombinant virus, or the mixture of such viruses, should be formulated with a suitable adjuvant to enhance the immunological response to their antigens. Suitable adjuvants include, but are not limited to, inorganic gels, for example aluminum hydroxide; surface-active substances such as lysolecithin, "Pluronic" polyols; polyanions; peptides; and emulsions in oil.

 Many methods can be used to introduce the vaccine formulations described above; they include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous and intranasal routes. 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 recombinant virus in the vaccine formulation.

FORMULATION OF SUBUNIT VACCINES
In lieu of virus-based vaccines, the LAV / HTLV III-related protein may be used as an immunogen in subunit vaccine formulations. As previously explained, subunit vaccines comprise only the relevant immunogenic material required to immunize a host. Thus, LAV / HTLV III-related protein can be purified from recombinants that express LAV epitopes.
HTLV III. Such recombinants include any of the virus-infected cultured cells, bacterial transformants, or yeast transformants that express the LAV / HTLV III epitopes (reference may be made to above about the insertion of protein coding sequences, the identification of recombinant expression vectors, and about the identification of the 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 hereinafter. above about the identification and purification of the expressed gene product).

Whether the immunogens are purified from recombinants or obtained by chemical synthesis, the final product can be adjusted to an appropriate concentration and formulated with any suitable vaccine adjuvant, and then packaged for use. Suitable adjuvants include, but are not limited to, inorganic gels, for example aluminum hydroxide, surfactants such as lysolecithin, polyols, and the like.
Pluronic; polyanions; peptides; and emulsions in oil. The immunogen may also be incorporated into lyposomes, or conjugated to polysaccharides and / or other polymers for use in a vaccine formulation.

When the peptide or protein related to LAV /
HTLV III is a hapten, that is a molecule that is antigenic because it can react selectively with recognized antibodies, but is not antigenic because it can not trigger an immune response, the hapten may be covalently bound to an immunogenic carrier or molecule; for example, a large protein such as protein serum albumin will impart an immunogenic character to the hapten coupled thereto. The hapten-carrier can be formulated for use as a vaccine.

PASSIVE IMMUNITY AND ANTI-IDIOTYPIC ANTIBODIES
Instead of active immunization with virus or subunit vaccines, it is possible to provide short-term protection to a host by administering preformed antibody against an epitope of LAV / HTLV. III.

Thus, the vaccine formulations described above can be used to produce antibodies for use in passive immunotherapy. Human immunoglobulin is preferred in human medicine since heterologous immunoglobulin may elicit an immune response to its foreign immunogenic components. Such passive immunization could be used in case of emergency for immediate protection of non-immune persons exposed to special risks, eg persons exposed to contact with AIDS patients, for example, in hospitals and other facilities. Alternatively, these antibodies can be used to produce antiidiotypic antibody, which in turn can be used as an antigen to stimulate an immune response against LAV / HTLV III epitopes.

IMMUNOLOGICAL ASSAYS
In another aspect of the present invention, the LAV / HTLV III related peptides and proteins of the present invention can be used as antigens in immunoassays for the detection of LAV / HTLV III antibodies in various tissues and fluids. patients, as well as blood from blood banks and hospitals.

 The antigens of the present invention can be used in any immunoassay system known in the art, which includes, but is not limited to, radioimmunoassays, so-called "ELISA" assays, "sandwich" assays, precipitation reactions, gel diffusion precipitation assays, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, immunofluorescence assays, immunoassays for protein A and assays by immunoelectrophoresis, just to name a few.

EXAMPLES
In the following examples, various plasmid vectors containing chimeric genes comprising downstream LAV envelope coding sequences were constructed relative to the vaccinia virus transcriptional control sequences.

 These chimeric genes containing the vaccinia promoter and the LAV envelope coding sequence were inserted into the genome of the vaccinia virus by in vivo recombination. Such recombinant viruses have been identified and purified, and virus stocks have been prepared from infected tissue culture cells. Immunoreactive proteins related to the LAV envelope have been shown to be produced by these viruses. recombinant vaccinia virus in vitro. These recombinants were tested in experimental animals for their ability to elicit neutralizing or protective immune responses and to study their use as AIDS vaccines. A detailed description of each step of the invention is provided. this embodiment of the invention is presented below.

 Here are some details about the general procedures.

CELLS AND VIRUSES
From R. Condit (Associate Professor,
Department of Biochemistry, State University of New York,
Buffalo, New York, USA) African green monkey kidney cells (strain BSC-40, continuous cell line of African green monkey cells
BSC-1, ATCC CCL26), and propagated in Dulbecco's modified Eagle's medium (MEMD, Gibco,
Grand Island, New York) supplemented with 10% fetal bovine serum and 100 penicillin units and 100 units of streptomycin per milliliter. Dr. Bachchan (Professor, Department of Molecular Biology, University of California, Berkley, California, United States of America) obtained human cells 143 TK t3 S. Rhim et al. , 1975,
Intl J. Cancer 15: 23-29) and was propagated in the above medium with the addition of 5-bromo-deoxyuridine (BUdR) at 25 μg / ml.

Raccid viruses (strain WR, ATCC VR-119) grown in BSC-40 cells in MEMD + 5% calf serum without gamma globulin + 100 units were obtained from R. Condit. / ml penicillin and 100 units / ml 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 gelose (DIFCO,
Detroit, Michigan, USA), 5% gamma-free globulin serum, 100 units / ml penicillin and 100 units / ml streptomycin and 25 μg / ml BUDR.

Dilutions of the virus stocks in phosphate buffered saline containing 8 g of NaCl, 0.2 g of KCl, 1.5 g of NaH 2 PO 4, 0.2 g of K 2 HPO 4 were made per liter with the addition of 1 mM MgCl2 and 0.01 X bovine serum albumin.

PREPARATION, RESTRICTION AND MODIFICATION OF DNA
Unless otherwise specified, all methods used for the following procedures are as described in the pages indicated by Maniatis et al., 1982, "Molecular Cloning", Cold Spring Harbor Laboratory: Plasmid DNA Preparation (pages 86-96). ), restriction digestion of DNA (pages 98-106) and purification of restriction fragments from low melting temperature agar gels (pages 157-161 and 170), reaction conditions for 1 DNA polymerase enzyme E. coli Klenow fragment (pages 107-114), alkaline phosphatase reactioP of the calf intestine (pages 133-134) and ligase reaction (page 146), preparation of well-translated probes (pages 109-112). ) and procedures for DNA-DNA hybridization (pages 324-325).

CONSTRUCTION OF PLASMID VECTORS CONTAINING THE VIRUS OF
VACCINE, PROMOTER, LINKED BY LIGATION AT THE SEQUENCES OF
ENCODING THE GENE OF THE LAV ENVELOPE
The following is described the construction of various plasmid vectors containing LAV envelope gene coding (adenopathy) sequences preceded by vaccinia virus transcriptional control sequences; these chimeric frequencies are surrounded by TK DNA.

These recombinant plasmid vectors were subsequently used to recombinantly invert the LAV envelope coding sequences into the vaccinia virus genome.

The coding sequence of the LAV envelope was purified from pRS-3, which is a subclone of lambda
J19 (S. Wain-Hobson et al., 1985, Cell 40: 9) and inserted into plasmid pGS20 (M. Mackett, GL Smith et al.
B. Moss, 1984, J. Virol. 49, 857-864) downstream from the vaccinia 7.5K promoter contained in pGS20, to construct pv-env, pv-env2 and pv-env5. In each of these constructs, the chimeric gene (i.e., the 7.5 K vaccinia promoter ligated to the LAV-specific nucleotide sequence) is surrounded by vaccinia TK gene sequences.

As previously explained, PRS-3 consists of a 3840 base pair fragment, from EcoRI to SstI, of the LAV DNA sequence contained by insertion into lambda
J19 cloned into the EcoRI and SstI site of pUCi8. This was achieved by cloning the J19 SstI restriction fragment introduced into the SstI site of pUC18 to create pBT-1> which was then digested with EcoRI and re-ligationed. This DNA was used to transform E. coli and plasmid pRS-3 was isolated. The LAV-specific DNA, contained in pRS-3, ranges from the EcoRI site, hosted at nucleotide 5289, to the SstI site located at nucleotide 9129 on the LAV genome (S. Wain-Hobson et al., 1985, Cell 40: 9); see Figure 2).

CONSTRUCTION OF PLASMID VECTORS CONTAINING VIRUSES OF
PROMOTER VACCINE CONNECTED BY LIGATION TO THE SEQUENCE ENCODING
IN 3 'OF THE GENE OF THE ENVELOPE OF LAV
Five micrograms of pRS-3 plasmid DNA were digested to completion with the restriction enzyme KpnI, and the resulting fragments were resolved on a low temperature 1% agar gel. fusion.

A fragment of 2.68 pKb (kilobase bases) was isolated and purified. This fragment contained the specific DNA of
LAV ranging from nucleotide number 5889 to 8572 and including sequences (5889-8349) that encode the terminal carbon portion of the LAV envelope protein.

 1 μg of this fragment was mixed with 0.5 μg of pGS 20 DNA which had been previously linearized with the BamHI restriction enzyme. Ligation of these two fragments was allowed to continue for 24 hours at 4 ° C in the presence of oligodeoxynucleotide linkers consisting of 0.6 μg of 5'-GATCCACCATGGTAC-3'-OH and 0.3 μg. These binding agents were used (a) to transform the KpnI sticky ends of the 2.68 pKb fragment from the pRS-3 plasmid DNA into cohesive ends of pRS-3. BamHI which are complementary to the BamHI cohesive ends of cleaved pGS DNA and (b) to provide a translation initiation sequence (ATG) in the correct reading frame of the LAV envelope gene sequence as well as nucleotide sequences necessary for efficient translation of the transcribed mRNA. The ligation mixture was used to transform E. coli strain MC1000. Plasmid DNA from ampicillin-resistant transformants was submitted. to tests for determining the orientation of the sequence ins SOE and regeneration BamHI sites, NcoI and KpnI the ligation junction. The confirmed structure of the desired plasmid, pv-env2, is shown in FIG. 3 in which the carboxylic coding portion of the LAV envelope gene corresponding to nucleotide numbers 58898672 (as shown in FIG. 2) is located downstream. compared to the 7.5 K vaccinia promoter. The LAV envelope sequence is placed in the correct reading frame with respect to the initiation ATG provided by the binding DNA.

CONSTRUCTION OF PLASMID VECTORS CONTAINING
LIGATION RELATED VACCINE VIRUS PROMOTER
AT THE SEQUENCE ENCODING 5 'OF LAV ENVELOPE GENE
Five micrograms of plasmid DNA from pRS-3 were digested to completion with restriction enzyme AvaI and the resulting fragments were resolved on a 1% gelose gel at a low temperature. fusion.

A fragment of 0.82 pKb was isolated and purified. This fragment contained LAV-specific sequences between nucleotide numbers 5671 and 6490 (as shown in Figure 2). This includes sequences (nucleotide numbers 57666490) encoding the nitrogenous terminal portion of the coat protein and 95 base pairs of non-translated LAV sequences close to 5 '. This fragment was treated with E. coli Klenow fragment DNA polymerase in the presence of an excess of deoxyribonucleotide triphosphate. The resulting blunt-ended fragment was ligated onto 0.5 μg of pGS 20 DNA which had been linearized with SmaI and treated with alkaline phosphatase from calf intestines (alkaline calf-intestine). phosphatase (CIAP)) The ligation was allowed to continue for 16 hours at 12 ° C. The ligation mixture was used to transform the E. coli strain MClGOO Plasmid DNA from resistant transformants was submitted. ampicillin to assays for determining the orientation of the LAV-inserted sequences, relative to the vaccinia virus transcriptional control sequences, The structure confirmed for the desired plasmid, pv-env, is presented on the FIG. 4, in which the coding amino portion of the LAV envelope gene, containing its own ATG initiation sequence, corresponding to nucleotides 6671 to 6490 (as shown in FIG. 2), is located e downstream with respect to the 7.5 K vaccinia promoter.

CONSTRUCTION OF PLASMID VECTORS CONTAINING VIRUSES
OF PROMOTER VACCINE CONNECTED BY LIGATION TO THE WHOLE
ENCODING SEQUENCES OF THE LAV ENVELOPE GENE
2 micrograms of plasmid pv-envl DNA were digested until completion with the restriction enzyme
StuI, PvuI and XhoI. The resulting fragments were resolved on a 1 x low melting temperature agar gel.

The 4 pKb fragment, containing the vaccinia virus transcription control element and the 5 'portion of the LAV envelope coding sequences, was isolated and purified.

This fragment was ligated to a 6.5 pKb fragment generated by pv-env2 digests using restriction enzymes StuI and PvuI. The ligation mixture was used to transform E. coli strain Mi 1000.

Ampicillin-resistant transformants were selected. 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 coat envelope gene.
LAV corresponding to nucleotides Nos. 5766 to 8349 (as shown in Figure 2), ligated downstream of the vaccinia virus transcription control elements.

CONSTRUCTION OF RECOMBINANT VACCINE VIRUS
CONTAINING THE CORRESPONDING CHIMERIC GENE
A LAV ENVELOPE
The insertion of the chimeric gene sequences corresponding to the envelope of LAV (LAV-env) into the genome of the vaccinia virus is obtained by "in vivo" recombination made possible by the fact that the chimeric genes are surrounded in the plasmids pv-env2 and pv-env5 by vaccinia virus sequences encoding the thymidine kinase (TK) gene. 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. Insertion of the chimeric gene occurs as a result of double recombination in the side surround sequences. Such recombinants will include the chimeric gene inserted into the vaccinia TK gene and, therefore, their phenotype will correspond to TK-. These TK recombinants can be selected for growth in a medium to which BUdR has been added, which is lethal for TK + cells but not for TK- cells. The general principle of this procedure has been described (M. Mackett, GL Smith and B. Moss, 1984, J. Virol.

49, 857-864).

A 100 ml diameter dish containing 80% confluent African green monkey kidney cells (strain BSC-40) was infected with vaccinia virus (strain
WR) at a multiplicity of infections of 0.05. After 2 to 4 hours of incubation at 370 ° C, the infected cells were coated with coprecipitates, using calcium phosphate, with plasmid pv-env2 or pv-env5 DNA. adding drop 0.5 ml of a 2XADN-CaCl 2 solution to 0.5 ml of 2XHeBS solutions (the 2XADN-CaCl 2 solution contains 20 μg of plasmid DNA in 0.5 ml of 0.25M CaCl 2; 2XHeBS contains, per ml, 16 mg NaCl, 0.74 mg KCl, 0.25 mg Na2HPO4 2H20, 2 mg dextrose, and 10 mg HEPES, at pH 7.08 was allowed to form at room temperature. The coprecipitates obtained with calcium phosphate-DNA were immersed for 30 minutes. Four hours after having covered them 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, and then washed again with 10 ml of growth medium (MEMD + 10% fetal calf serum + 100 units / ml of penicillin line and 100 units / ml of streptomycin). Two days later, the infected cells were harvested and collected by centrifugation (4 C, 10 minutes at 2000 xg). Virus stocks were prepared by resuspending these cells in 1 ml of phosphate buffered saline and addition of bovine serum albumin followed by two cycles of freezing and thawing and three treatments. for 15 seconds by 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 ml tK confluent human cells, topped with 5 ml / well. 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 ml) virus suspensions to infect TK confluent cells placed in 16 mm diameter depressions in a selective medium (MEMD + 10% fetal calf serum + 100 units / ml streptomycin + 100 units / ml of penicillin + 25 g / ml of
BUdR). The infected cells were collected by centrifugation and resuspended in 100 ml 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 of 20 seconds each of sonication. The cell lysates were collected on nitrocellulose filters, using a multi-trough manifold or manifold (Schleicher and Schuell,
Arlington, MA). DNA-DNA hybridization was determined as described (M. Mackett, GL Smith and B. Moss, 1982, Proc.

Nat'l. Acad. Sci. 79, 7415-7419) the presence of LAV-specific DNA sequences in these samples. The 32 P-labeled plasmid pRS-3-DNA hybridization probe was 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 plates on BSC-40 cells and used for subsequent characterization.

A schematic representation of the construction of the recombinant viruses is shown in Figure 5, in which the gene sequence corresponding to the LAV envelope (env) located downstream of the 7.5K vaccinia promoter (Par) is surrounded within the vaccinia genome by TK DNA sequences. Virus stocks from recombination between the vaccinia virus genome and the plasmid pv-env5 have been termed v-env5, and they contain the entire gene of the LAV envelope. In the particular embodiment described in the examples herein, v-env5 contains all of the coat gene as well as 96 base pairs of non-translated sequences close to 5 'and 223 base pairs of the near non-translated sequences. The virus stocks from pvenv2 were called v-env2 and they contain most of the LAV envelope gene (ie the fragment
KpnI), but they do not include the portion of the sequence encoding the first 42 amino acids of the LAV envelope protein. Since the supposed signal sequence of the LAV envelope protein is located within the first 49 amino acids of the protein, v-env2 should produce a protein lacking the signal sequence; therefore, it should be expected that the LAV-related protein produced by this recombinant virus will not be transported to the membrane. In contrast, v-env5, which contains the entire LAV envelope sequence, must produce a protein that is transported to the membrane.

EXPRESSION OF PROTEINS RELATED TO LAV ENVELOPE
IN INFECTED TISSUE CULTURE CELLS
BY RECOMBINANT VACCINE VIRUSES
Recombinant vaccinia viruses, carrying chimeric LAV envelope genes, have been shown to be capable of expressing the envelope-related proteins.
LAV during cell infection in tissue culture. It has also been found that these proteins are immunoreactive with serum from patients with
AIDS.

-IDENTIFICATION, USING IMMUNO-TECHNIQUES
COAGULATION OF PROTEINS RELATED TO THE DEVELOPMENT OF LAV EXPRESSED IN CELLS INFECTED WITH VACCINE VIRUS
Recombinant.

 A circular dish plate of 100 ml of BSC40 confluent cells, at a rate or multiplicity of infection of 10, was infected with wild-type vaccinia virus or with its recombinant derivatives, v-env5 or v-env2.

The infection was allowed to continue for 12 hours, after which cells were harvested, washed once with physiological phosphate buffered saline (PBS) and collected by centrifugation.

The pellets of infected cells were resuspended in 1 ml of Laemmli sample buffer (Laemmli,
UK, 1970, Nature 227: 680) and was lysed for 4 minutes boiling. The total cellular protein in an aliquot (75 L) of cell lysate was resolved by electrophoresis on a SDS-polyacrylamide gel at a gradient of 7 to 15%. A virion sample of purified LAV and an aliquot of "infected" cell lysate were included as a control by a non-pathogenic (i.e., uninfected) virus. The gel contents were electroblotted onto a nitrocellulose filter sheet.

The filter was first incubated in 5 ml of PBS (phosphate buffered saline) plus 5% defatted milk powder, for 20 minutes at room temperature and then for 2 hours at room temperature in PBS. + 5% defatted milk powder + human serum from AIDS patients (1: 100 dilution of inactivated serum by heating). The filter was then washed five times with PBS + 0.05% "Tween 20" (polyoxyethylene sorbitan monolaurate) and once with
PBS alone. The filtrate thus washed with 5 ml of PBS containing 1% normal goat serum (inactivated by heating) plus a 1/3000 dilution of IgG conjugate (immunoglobulin) was incubated for 2 hours at room temperature.
G) anti-human goat / horseradish peroxidase. The same filter was washed 5 times with PBS + 0.05% Tween 20, and once with TBS (0.5M NaCl + 20mM Tris-HCl, pH 7.5). The horseradish peroxidase conjugate attached to the filter was detected by reacting chloronaphthol staining reagents with the filter for 10 minutes at room temperature in the dark (the chloronaphthol staining reagent was prepared by mixing solutions A and B just before use.
A: 20 m 1 of cold 30% methanol + 60 mg of 4-chloro-naphthol; solution B: 60 μl of cold hydrogen peroxide + 100 ml of TBS).

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

The results of this assay, as shown in Figure 6A, indicated that vaccinia virus V-env5 produced a family of three proteins that were able to exhibit specific immune reactivity with serum from patient with AIDS. These proteins had electrophoretic mobility similar to that of gap150, gp10, and gp41 glycoproteins of the authentic LAV / HTLV III virus, which is thought to be encoded by the envelope gene (Robey, W., et al., 1985).
Science 228: 593-595). These proteins have not been produced in cells infected with non-infectious virus or in cells infected with recombinant V-type wild-type vaccinia virus, which does not have the 5 'sequences coding for methionine. supposed initiation and the first 42 amino acids of the envelope proteins
LAV, produced a protein of small dimensions, but still immunoreactive with a serum of patient with AIDS. It is believed that translation of the env sequence in recombinant v-env2 starts from the AUG codon transcribed from the sequence corresponding to the binding agent used in the construction of this recombinant (see above the construction of plasmid vectors containing vaccinia virus ligated to the 3 'LAV envelope gene sequences).

 In a second experiment, two cell lines (BSC-40 and HeLa) were infected with v-env5 and v-env2.

The LAV-specific proteins in the infected cell lysates were subjected to Western immunocoagulation assays as described below.

Confluent cell monolayers have been infected
BSC-40 or HeLa with v-env5 or v-env2 recombinant virus at a multiplicity or infection rate of 50 plaque forming units per cell. Twelve hours after infection, the cells were washed twice in phosphate buffered saline, resuspended in Laemmli sample buffer, and boiled for 5 minutes. The proteins from infected cells were resolved by polyacrylamide gel electrophoresis on sodium dodecyl sulfate gel (SDS-PAGE), electroblotted to a nitrocellulose membrane and reacted with pooled serum from Serum positive in the LAV / HTLV-III search. Protein A, which was 125 I-labeled by the chloramine T method, detected immunoreactive proteins. In Figure 6B, lanes 1 and 5 contain proteins from cells infected with non-infectious viruses; lanes 2 and 6 contain proteins from cells infected with wild-type vaccinia virus; lanes 3 and 7 of proteins from v-env-5 infected cells; and lanes 4 and 8 are from cells infected with v-env2. LAV virion proteins purified using a sucrose gradient were used as a control (LAV), and positions of the gp150, gel 10 and gp41 envelope proteins were as shown in the figure.

Molecular weights were expressed in kilodaltons (kd).

 In v-env infected cells (FIG. 6B, lanes 3 and 7), three major proteins were identified that were capable of an immune response with pooled serum from serum individuals giving a positive response. Molecular weights of these proteins were estimated to be 150 kd, 120 kd, and 41 kd, similar to those of LAV gap glycoproteins gap150, gpl10, and gp41.

 V-env2 of recombinant virus did not have the sequence expected to signal for the LAV envelope, but could produce at least three immunoreactive polypeptides with molecular weight 99 kd, 68 kd and 40 kd (Figure 6B, lanes 4 and 8). ).

IDENTIFICATION, BY IMMUNOPRECIPITATION TECHNIQUES,
OF PROTEINS RELATED TO LAV ENVELOPE AND EXPRESSED
IN CELLS INFECTED WITH VACCINE VIRUS
Recombinant
The immunoprecipitation assay, described hereinafter, has shown that cells infected with recombinant vaccinia viruses of the present invention synthesize proteins capable of a specific immune response with serum from AIDS patients.

 Wild-type vaccinia virus, or its recombinants v-env5 or v-env2, were infected at a rate or multiplicity of infection of 10, a 100 ml dish containing confluent BSC-40 cells. After a lapse of 9.5 hours after infection, the growth medium was replaced by MEMD without methionine and without supplemental serum. At 10 hours after infection, the medium was replaced with 2 ml of methionine-free MEMD containing 100 μCi / ml of (35 S) methionine and the labeling was allowed to continue for 2 hours at 37 ° C. of the labeling period, cells were washed once with physiological phosphate buffered saline (PBS) and collected by centrifugation.

The cell pellets were resuspended in 1 ml of lysis buffer: 1% NP 40 (polyoxyethylene (9) p-tertoctylphenol), 0.5% sodium deoxycholate, 0.1 M NaCl, tris-HCl 0.01M, pH 7.4, 1mM EDTA, and the lysate was removed by centrifugation for 1 minute in an Eppendorf microcentrifuge.

Immunoprecipitation was performed by adding 5 microliters of inactivated human serum by heating from a control or AIDS patient population to 100 ml of cell lysate. After 1 hour of incubation at 4 ° C, 60 ml of activated Staphylococcus aureus cells ("Pansorbin" cells, Calbiochem-Behring Corp.,
LaJolla, CA) and the incubation was allowed to continue for an additional 1 hour at 4 ° C. The immunoprecipitation complexes were collected by centrifugation for 30 seconds in a 4 C Eppendorf microcentrifuge and washed. 1M NaCl + 0.1% NP 40 + 0.01M Tris-HCl, pH 7.4, and twice RIPA buffer (10mM Tris-HCl, pH 7.2, 0.15M NaCl, 1 M NaCl). % sodium deoxycholate, 1% "Triton-X 100" (polyoxyethylene (9-10) p-tert-octylphenol), 0.1% sodium lauryl sulphate) The immunoprecipitates, washed, were resuspended. 50 ml of a Laemmli sample buffer, boiled for 1 minute and centrifuged for 1 minute in a microcentrifuge
Eppendorf. The immunoprecipitated proteins present in the supernatant were analyzed by electrophoresis on SDS-15 polyacrylamide gels. After electrophoresis, the gels were stained with Coomassie blue stain, treated with sodium salicylate. (30 minutes at room temperature in 1M sodium salicylate) and dried for fluorography.

 The results of this assay, as shown in Figure 7A, indicated that recombinant v-env5 synthesized a family of proteins exhibiting specific immunoreaction with AIDS patient serum.

These proteins had apparent molecular weights of 160, 140, 120, 42 and 40 kilodaltons (kd), approximately corresponding to the apparent molecular weights of the envelope-related glycoproteins reported for LAV / HTLV III (WG Robey et al., 1985, Science 228: 593-595). These proteins were not present in cells "infected" with non-infectious viruses or in cells infected with wild-type vaccinia virus and they also did not undergo immunoprecipitation with human control serum. cells infected with v-env2, a truncated protein with an apparent molecular weight of 95 d, was produced and recognized by AIDS patient serum.

This truncated form of LAV envelope-related protein most likely proceeds from the AUG codon encoded by the 5 'linker sequence near the inserted sequence, corresponding to LAV, in this recombinant.

3H-GLUCOSAMINE MARKING OF PROTEINS RELATED TO
ENVELOPE OF LAV PRODUCED BY RECOMBINANT VIRUSES
OF VACCINE CORRESPONDING TO LAV
The radioimmunoprecipitation assay described hereinafter indicated that the vaccinia recombinant viruses, corresponding to LAV, of the present invention produced glycosylated coat proteins.

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

Aliquots of cell lysate were mixed with normal human serum (lanes 1-4) or pooled serum from LAV / HTLV III positive serum (lanes 5-8). Immunoreactive proteins were precipitated by Staphylococcus aureus fixed cells carrying protein A, then resolved by SDS-PAGE and fluorographic detection.

 The radioisotope-labeled glucosamine labeling results, as shown in Figure 7B, lane 7, indicate that the proteins obtained using the recombinant v-env5, such as LAV envelope proteins, were also glycosylated. Differences in glycosylation patterns may explain the slight variations observed in mobilities by electrophoresis of recombinantly produced proteins compared to LAV virion glycoproteins.

 As would be expected for proteins lacking signal-emitting peptides, nitrogen-linked glycosylation with 3H-glucosamine was not observed in v-env2 infected cells (FIG. 7B). track 8).

IMMUNOPRECIPITATION ANALYSIS, WITH HUNTING PULSE,
OF PROTEINS RELATED TO THE LAV ENVELOPE PRODUCED
BY RECOMBINANT VACCINE VIRUSES RELATED TO LAV
It has been suggested that gap150 of LAV is the precursor from which a gel outer protein and a gp40 transmembrane protein originate. The "hunting pulse" immunoprecipitation test described hereinafter indicates that the 150 kd, 120 kd and 41 kd proteins obtained using the recombinant vaccinia virus corresponding to
LAV, have a relation between precursor and product similar to that suggested for gap150, gpl10 and gp41 for authentic LAV / HTV III.

Confluent monolayers of HeLa cells were infected with wild type vaccinia virus (see Figure 7C, lanes 1-6), recombinant v-env (lanes 7-12) or v-env2 (lanes 13-18), the all at a multiplicity of infection of 50 plaque forming units / cell. After 10 1/2 hours after infection, the cells were labeled with 35 S-methionine (greater than 800 Ci / mmol, Amersham) at 100 pCi / ml for 15 minutes. At the end of the labeling period, the cells were washed. the cells once with 2 ml of prewarmed hunting medium modified Eagle's medium according to
Dulbecco, MEMD, + 3 mg / ml L-methionine + 5% calf serum, 100 units / ml penicillin + 100 μg / ml streptomycin) and then cells were again provided with 1 ml of the same medium before return them to the incubator.

At various times thereafter, the cells were washed and lysed as previously described above (H-glycosamine staining) and proteins from immunoprecipitation cell lysates were subjected to pooled serum from of people whose serum had a positive response for LAV / HTLV-III. Immunoprecipitated proteins were resolved by electrophoresis on
SDS-PAGE and detected by fluorography. The duration of each hunt was as follows: O hour (lanes 1, 7, 13); 1 half hour (tracks 3, 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). The results presented in FIG. 7C, lanes 7-12, indicate for the proteins at 150 kd, 120 kd and 41 kd obtained using the recombinant, the same precursor-product relationship as for gp150, gpl10 and gp41 of LAV / HTLV III authentic. Treatment of the 150 kd protein appeared slow and ineffective in HeLa cells, since after 6 hours cleared the pulse labeling, less than 50% of the 150 kd protein radioactivity was passed through the proteins. 120 kd and 41 kd. Preliminary results indicate that this treatment is more effective in certain types of human peripheral blood cells infected with the same recombinant viruses. These experiments also indicated that the env sequence in v-en2, which did not include the sequence supposed to emit a signal to detect the LAV envelope, was expressed as an unmodified 87 kd precursor (780 amino acids), which yielded a 99 kd intermediate and was cleaved into the 68 kd and 40 kd polypeptides. (Figure 7C, lanes 13-18).

PRESENCE OF A PROTEIN RELATED TO THE LAV ENVELOPE
IN THE GROWTH ENVIRONMENT OF CELLS INFECTED BY
RECOMBINANT VACCINE VIRUSES LAV
The radioimmunoprecipitation assay described hereinafter showed that the immunoreactive proteins produced by the recombinant vaccinia virus of the invention can be expressed and treated similarly to the authentic LAV envelope glycoprotein.

Confluent monolayers of HeLa cells infected with non-infectious virus (see Figure 7D, lane 1) or separately infected with wild-type vaccinia virus (lane 2) were submitted with recombinant v-env5 (lane 3 ) or recombinant v-env2 (lane 4), all at a
multiplicity or infection rate of 20 plaque forming units per cell. The cells were labeled with ethionine (greater than 800 Ci / mmol, Amersham) at 100 pCi / ml at 12 hours post infection. At the end of the labeling period, the medium was removed and clarified by centrifugation for 2 minutes at 12,000 g prior to use for pooled serum immunoprecipitation from persons with a positive result. LAV / HTLV III.Proteins from infected cells immunoprecipitated with the same serum are presented in the portion marked "pellet" and the proteins from the growth medium or culture are presented in the portion marked "Supe" (upper medium). supernatant).

 As expected for LAV, the 120 kd protein obtained with the recombinant was also detected in the infected medium (Figure 7D, lane 3). These results showed that recombinant virus env-env5 was able to express the gene corresponding to the LAV envelope and produce immunoreactive proteins that were treated similarly to authentic LAV envelope glycoproteins.

 As expected for proteins lacking signal peptides, no LAV envelope-related polypeptides were detected in the growth medium of v-env2 infected cells (FIG. 7D, lane). 4).

IMMUNE POWER OF THE RECOMBINANT VIRUS
CORRESPONDING TO THE ENVELOPE OF LAV
Recombinant viruses, carrying the chimeric gene corresponding to the LAV envelope, have been shown to be capable of eliciting an antibody response to LAV in two mouse strains and one subhuman primate species.

IMMUNOGENICITY OF RECOMBINANTS OF VACCINE CARRYING
THE ENVELOPE GENE OF LAV IN MOUSE
Immunogenicity of proteins expressed by v-env5 and v-env2 of recombinant vaccinia viruses was examined. The experiments described hereinafter indicate the ability of these recombinant viruses to elicit an immune response for all major LAV glycoproteins.

 In two mouse lines, one inbred (C57B16J) and the other non-consanguineous (ICR), v-env2 or v-env5 were inoculated with recombinant viruses. All animals were 5-7 weeks old at the time of inoculation. Four inoculation routes were used: pad, paw-, tail scarification, intranasal and intraperitoneal routes. Inoculation was performed in the paw pad by injecting 25 μl (5 × 10 6 plaque forming units) of recombinant virus into each of the pads of the hind paws. Tail scarification was performed by scraping the skin at the base of the tail with a two-pointed needle and applying 10 μl (2 x plaque forming units) of the virus to the scarred surface. Recombinant. Intranasal inoculation was performed by placing 10 μl (2x10 7 units of plaque formation) of recombinant virus on the nose of the mice and allowing the mouse to aspirate the inoculum. Intraperitoneal injections were performed by injecting 10 μl (2 x 10 units of plaque formation) of recombinant virus into the peritoneal cavity of the mice.

The stock preparation and virus dilution were performed by operating as described above with respect to cells and viruses. Serum samples were collected at 2 week intervals after inoculation into individual mice and analyzed by an enzyme-linked immunosorption assay (ELISA'I enzyme linked immunosorbent assay) and by Western coagulation analysis as described below.

SERUM CONVERSION (SEROCONVERSION), SHOWN BY
THE ELISA ASSAY OR ASSAY OF IMMUNIZED MOUSE
BY RECOMBINANT VIRUSES OF CHIMERIC GENE VACCINE
LAV ENVELOPE
The results of the ELISA tests on 6-week serum samples are summarized in Table 1. The recombinant virus v-env2 and v-env5 sequences converted (transformed) to 100% and 95% the serum of the C57B16J mice, respectively . For ICR mice, the serum conversion rate was 44% for v-env2 and 100% for v-env5.

Total serum conversion was achieved as well as the highest average titre in the ELISA assay in mice immunized by tail scarification.

TABLE I
Seroconversion of mice inoculated with viruses
recombinants carrying the chimeric gene of
the envelope of LAV
Recombinant virus Inoculation route Mouse seroconversion
inoculated *
ICR C57B16J v-env2 foot pad 1/3 4/5
Tail Scarification 3/3 4/5
Intranasal route 0/3 4/5
Intraperitoneal route not performed 3/5 v-env5 Paw pad 4/4 5/5
Tail Scarification 3/3 5/5
Intranasal route 3/3 4/5
Intraperitoneal route not performed 5/5 * Seroconversion, determined by ELISA as
described, is expressed by the number of mice
seroconversion to the total number of mice
having been inoculated, for each group.

 The results of the ELISA assays or assays were obtained as follows: inactivated and purified LAV virions were diluted in carbonate buffer (50 nM sodium carbonate, pH 9.6) and dilutions in microtiter plates with 96 wells or wells (100 Al / well, containing 0.2 μg of inactivated LAV). Binding was allowed to proceed at 4 ° C overnight. The unbound protein was aspirated and washed with 200 ml (by plating) of phosphate buffered saline + 5% defatted milk powder and then with 300 μl (per trough) at 4% sucrose After suctioning of the excess sucrose solution, the plates were allowed to dry at room temperature (1 hour), then 50 μl of phosphate buffered saline containing 2.5 μl of mouse serum sample and allowed to react at 37 ° C for 1 hour. At the end of the incubation period, the wells were washed five times with physiological saline solution containing phosphate buffer (PBS) + 0.05% "Tween 20" (polyoxyethylene sorbitan monolaurate). 50 μl of goat anti-mouse immunoglobulin G / horseradish peroxidase conjugates (1: 5000 dilution in PBS (physiological phosphate buffered saline) + 0.05% Tween-20 were added to each well, and allowed to react at 37 ° C for 45 minutes.

After 5 washes with PBS + 0.05% Tween 20, 100 μl of a citrate-O-phenylenediamine-peroxide substrate was added.

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

 The results shown in Table I are expressed as ratios of the total number of seropositive mice to the total number of mice inoculated.

Serum samples were collected 6 weeks after inoculation and analyzed by the ELISA assay. Serum samples from 5 non-inoculated mice were used as control. Mean ELISA titers for the control group were 0.021 + 0.005 and 0.017 + 0.010 for the ICR and C57B15J mice, respectively. Serum samples from immunized inoculation mice were considered positive and showed ELISA titers more than three standard deviations from the control group.

To show that mice inoculated with recombinant viruses ("seroconverted" mice) produced antibodies against authentic LAV envelope glycoproteins, serum samples from mice that were inoculated ("immunized mice") were analyzed. ") by a Western immunocoagulation technique as follows:" inactive "male mice 5 to 7 weeks old, consanguineous (C57B16J, Jackson
Laboratory) by scarification of the tail, which involved applying a 10 1 inoculum containing 2x107 recombinant vaccinia virus plaque forming units to abrasions generated by scraping with a two-pointed needle. at the base of the tail. The animals were bled from the retro-orbital sinus 8 weeks after inoculation and the serum was kept frozen until use. The aliquots of serum samples, diluted 50-fold in phosphate buffered saline + 0.2% NP-40 and 3% non-fat milk powder, were reacted with virion proteins. LAV that have been resolved by polyacrylamide electrophoresis
SDS and immobilized by electrotransfer on nitrocellulose filters. Alkaline phosphatase-conjugated goat anti-mouse immunoglobulin was detected by the LAV proteins recognized by these sera.

 Results obtained on 8 week old serum samples from inoculated C57B16J mice, by tail scarification, of recombinant viruses are shown in Figure 8. All animals inoculated with recombinant viruses produced antibodies. which reacted with the glycoprotein gp41 of the LAV envelope. Serum from some of the animals inoculated with v-env5 (Figure 8, lane a) also recognized gp50 and gp10, indicating the ability of this recombinant virus to elicit an immune response to all major glycoproteins of LAV / HTLV III.

IMMUNOGENIC CHARACTER OF RECOMBINANT V-ENV5
VACCINE VIRUS IN SUB-HUMAN PRIMATES
Five long-tailed macaques were used (Macaca
Fascicularis), ranging from youth to adulthood, to study the immunogenic character of vaccinia virus recombinant v-env5. All animals were screened for the absence of antibodies to vaccinia virus and LAV and the absence of virus.
T-lymphotropes of monkey or monkey AIDS virus or antibodies to these viruses. To four of the monkeys, 2 x 10 8 v-env5 plaque formation units were inoculated and 2 x 107 plaque forming units of a recombinant vaccinia virus (v-HSgD1 of recombinant virus) were inoculated into a monkey. Herpes simplex gD1), all by scarification of the skin. Serum and whole blood samples were collected prior to inoculation and after 3, 4 and 6 weeks after inoculation. All animals exhibited self-healing of skin lesions typical of vaccinia virus infections and normal physiological results throughout the course of the experiment. The humoral and cell-mediated immune responses were analyzed by Western immunocoagulation assay and proliferation assays, respectively.

HUMORAL IMMUNE RESPONSE, MONTREE
BY AN IMMUNOCOAGULAT TEST OF WESTERN, IN MACAQUES
Serum samples were collected prior to inoculation and after 3.4 and 6 weeks after inoculation.

Serum aliquots were diluted 50-fold and reacted with resolution-resolved SDS virion protein on SDS-polyacrylamide gel and immobilized by electrotransfer on nitrocellulose filters. The LAV proteins recognized by these sera were detected by goat anti-human immunoglobulin, conjugated with alkaline phosphatases. Grouped sera from seropositive individuals for LAV / HTLV III (AIDS) were used as a positive control.

 The results of Western coagulation analysis on macaque serum samples are shown in Figure 9. Of the four monkeys, three (monkeys # 168, 175, and 180) produced, as of the third week after Inoculation of antibodies to LAV envelope glycoprotein gp41 Monkey 182, which did not show a detectable level of response to LAV, also had a lower serum neutralization titre against vaccinia virus, compared with from the rest of the group.

IN VIVO SENSITIZATION OF T CELLS (LYMPHOCYTES) IN
MACAQUES VACCINATED WITH RECOMBINANT VACCINE VIRUS
A CHIMERIC ENVELOPE GENE
Picole peripheral macaque blood lymphocytes were isolated by centrifugation 6 weeks after vaccination and suspended at 1 × 10 6 lymphocytes / ml in RPMI 1640 medium containing 10% inactivated normal human serum. 0.1 ml of sensitive peripheral blood lymphocytes were cultured in 96-well plate wells, with total absence of antigen, with vaccinia virus recombinant, chimeric envelope gene, ultraviolet (UV) inactivated or UV inactivated starter vaccine (1 x 106 units of plaque formation per ml before UV inactivation), then 7-days later the proliferation was determined T-cells by determining the incorporated H3-thymidine during the last 6 hours of a 7-day culture.

 The results presented in Table II indicate that macaque peripheral blood T lymphocytes show, 6 weeks after vaccination, significant proliferative responses after in vitro stimulation with starting vaccinia virus or with recombinant chimeric gene virus v-env5. In addition, the response was similar in magnitude to that of the 173 macaque cells, which was. vaccinated with recombinant vaccinia virus v-HSgD1 at herpes simplex gD. These results indicate that the expression of the LAV envelope gene by the recombinant virus in macaques did not suppress in vitro or in vivo the T-cell mediated immune responses.

TABLE II
In vivo "sensitization" of T cells in vaccinated macaques
by the recombinant vaccinia virus with envelope gene ("vaccin-env" 0
Incorporation of H-thymidine (strokes per minute)
Macaque Vaccination with lymphocyte cells after 7 days of culture n No antigen Vaccine from start-up vaccin-env 168 vaccin-env 520 36 755 36 942 175 vaccin-env 7 640 36 405 35 142 180 vaccin-env 3 962 32 960 30 237 182 vaccin-env 929 11 077 12 692 173 vaccinia-herpes simplex gD 1 757 35 837 31 860 208 none 6 102 4 867 9 420 233 none 4 100 4 755 5 530
Overall, the results shown in Figures 6-9 and Tables I and II indicate that the recombinant vaccinia viruses described herein were able to (a) express the inserted LAV envelope gene (b). ) to modify or treat gene products to approximate authentic virus-related LAV proteins, (c) to produce immunogenic proteins with specific immunoreactivity with AIDS patient serum, (d) to trigger specific responses to LAV (lymphadenopathy) in mice and subhuman primates; and (e) the recombinant virus has not suppressed immune responses, mediated by T cells in vitro or in vivo.
DEPOSIT OF MICROORGANISMS
The following strains of E. coli, carrying the enumerated plasmids, were deposited at Agricultural Research
Culture Collection (NRRL), Peoria, Illinois (United States of America) and have received the following registration numbers ::
E. coli Strain Plasmid Registration number
K 12 Mi 1000 pv-env NRRL B-18003
K 12, MC1000 pv-2 NRRL B-18004
K 12, Mi 1000 pv-env5 NRRL B-18005
The following recombinant viruses have been deposited at the American Type Culture Collection (ATCC), Rockville, MD.
United States of America, and the following deposit numbers have been received
recombinant virus deposit number
v-env2 ATCC VR 2114
v-env5 ATCC VR 2113
It should not be considered that the scope of the present invention is limited only to deposited microorganisms and viruses, since what is deposited is a mere illustration of one aspect of the invention and that all microorganisms or viruses functionally Equivalents are within the scope of the present invention. It goes without saying that without departing from the scope of the invention, many modifications can be made to recombinant viruses, their chimeric genomes and proteins or peptides and vaccine formulations, described and represented.

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

Claims (61)

 A recombinant virus characterized in that its genome comprises a nucleotide sequence coding for an adenopathy-associated virus epitope (LAV) / leukemia virus of human T cells or lymphocytes (HTLV-III), or a antigenic portion of this sequence which is under the control of a second nucleotide sequence which regulates the expression of the gene so that a peptide, or a protein related to the LAV / HTLV III epitope is expressed in a host infected with the virus.  2. The recombinant virus as claimed in claim 1, characterized in that the nucleotide sequence coding for the LAV / HTLV III epitope comprises the sequence of the LAV / HTLV III envelope gene or any part of this coding gene. for an antigenic peptide or protein.  Recombinant virus according to claim 2, characterized in that the nucleotide sequence of the coat gene comprises the nucleotide sequence as shown in Figure 2, from nucleotide number 6767 to 8549, or any part thereof. of this sequence encoding an antigenic peptide or an antigenic protein.  4. The recombinant virus according to claim 1, characterized in that the peptide or protein expressed in the infected host is bound to a coat envelope epitope. LAV / HTLV III, or any antigenic part thereof, or the peptide or expression protein.  Recombinant virus according to claim 4, characterized in that the envelope epitope comprises a peptide or a protein having an amino acid sequence substantially as shown in Figure 2 from the amino acid residue number 1 to the residue number 361, or any antigenic part of this sequence.  6. Recombinant virus according to any one of claims 1 to 5, characterized in that it comprises a virus comprising an envelope.  Recombinant virus according to claim 6, characterized in that it is or comprises a vaccinia virus.  8. v-env5 sequence of vaccinia virus as deposited at the American Type Culture Collection (ATCC) and which has been assigned accession number VR 2113, or a mutant, a recombinant or a derivative of this genetically engineered virus genetic.  9. v-env2 sequence of vaccinia virus, such as this virus is deposited at the American Type Culture Collection (ATCC) and has been assigned the accession number VR 2114 or a mutant, recombinant or derived from this genetically engineered virus genetic.  10. The recombinant virus according to any one of claims 1 to 5, characterized in that it comprises a naked virus.  11. The recombinant virus according to claim 10, characterized in that it is or comprises an adenovirus.  12. The recombinant virus according to any one of claims 1 to 5, characterized in that it comprises a nuclear polyhedrosis virus.  13. The recombinant virus according to claim 12, characterized in that it is or comprises a baculovirus.  14. The recombinant virus according to any one of claims 1 to 5, characterized in that it is or comprises a bacteriophage.  15. Peptide or essentially pure antigenic protein, characterized in that it is related to an epitope of LAV / HTLV III.  Peptide or protein according to claim 15, characterized in that the epitope comprises an envelope glycoprotein of LAV / HTLV III.  Peptide or protein according to claim 15, characterized in that it comprises a sequence of amino acids comprising the amino acid sequence as described in Figure 2, from the amino acid residue number 1 to the remainder 861, or any antigenic part of this sequence.  Peptide or protein according to any one of claims 15 to 17, characterized in that this peptide or protein has been purified from a cultured cell containing a nucleotide sequence coding for the peptide. or the protein that is under the control of a second nucleotide sequence regulating gene expression, so that the peptide or protein is expressed by the cultured cell.  Peptide or protein according to claim 18, characterized in that the cultured cell is or comprises a microorganism.  Peptide or protein according to claim 19, characterized in that the microorganism is or comprises a bacterium.  Peptide or protein according to claim 19, characterized in that the microorganism is or comprises a yeast.  Peptide or protein according to claim 18, characterized in that the cultured cell is or comprises an animal cell line.  23. Peptide or protein according to claim 18, characterized in that the cultured cell is or comprises an insect cell line.  Peptide or protein according to any one of claims 15 to 17, characterized in that the peptide or protein has been obtained by chemical synthesis.  25. Formulation of live virus vaccine, characterized in that it comprises a recombinant virus according to any one of claims 1 to 5, the virus being infectious without causing disease for the host to be vaccinated.  26. Live virus vaccine formulation according to claim 25, characterized in that the virus is or comprises an enveloped virus.  27. Live virus vaccine formulation according to claim 20, characterized in that the enveloped virus is or comprises a vaccinia virus.  28. Live virus vaccine formulation, characterized in that it comprises an infectious dose of the vaccinia virus v-env5 gene sequence according to claim 8.  29. Live virus vaccine formulation characterized in that it comprises an infectious dose of the vaccinia virus v-env2 gene sequence of claim 9.  30. Live virus vaccine formulation according to claim 25, characterized in that the virus is or comprises a naked virus.  31. Live virus vaccine formulation according to claim 30, characterized in that the naked virus is or comprises an adenovirus.  32. Formulation of inactivated virus vaccine, characterized in that it comprises an effective dose of the recombinant virus according to any one of claims 1 to 5, in a non-infectious state, in admixture with a pharmaceutical excipient.  33. Formulation of inactivated virus vaccine characterized in that it comprises an effective dose of the recombinant envelope virus according to claim 6, in a non-infectious state, in admixture with a pharmaceutical excipient.  34. Formulation of inactivated virus vaccine characterized in that it comprises an effective dose of the vaccinia virus of claim 7, in a non-infectious state, in admixture with a pharmaceutical excipient.  35. Formulation of inactivated virus vaccine characterized in that it comprises an effective dose of the v-env5 sequence of vaccinia virus according to claim 8, in a non-infectious state, in admixture with a pharmaceutical excipient.  36. An inactivated virus vaccine formulation, characterized in that it comprises an effective dose of the v-env2 sequence of the vaccinia virus according to claim 9 in a non-infectious state, in admixture with a pharmaceutical excipient.  37. An inactivated virus vaccine formulation, characterized in that it comprises an effective dose of the naked virus according to claim 10, in a non-infectious state, in admixture with a pharmaceutical excipient.  38. Formulation of inactivated virus vaccine, characterized in that it comprises an effective dose of the adenovirus of claim 11, in a non-infectious state, and a mixture with a pharmaceutical excipient.  39. Formulation of inactivated virus vaccine, characterized in that it comprises an effective dose of the nuclear polyhedrosis virus according to claim 12, in a non-infectious state, in admixture with a pharmaceutical excipient.  40. Formulation of inactivated virus vaccine, characterized in that it comprises an effective dose of baculovirus according to claim 13, in a non-infectious state, in admixture with a pharmaceutical excipient.  41. Formulation of inactivated virus vaccine, characterized in that it comprises an effective dose of the bacteriophage of claim 14 in a non-infectious state, in admixture with a pharmaceutical excipient.  42. A subunit vaccine formulation, characterized in that the immunogen comprises an effective dose of the peptide or protein of any one of claims 15, 16 or 17, in admixture with a pharmaceutical excipient.  43. A subunit vaccine formulation characterized in that the immunogen comprises an effective dose of the peptide or protein of claim 18, in admixture with a pharmaceutical excipient.  44. A subunit vaccine formulation, characterized in that the immunogen comprises an effective dose of the peptide or protein of claim 199 in admixture with a pharmaceutical excipient.  45. A subunit vaccine formulation, characterized in that the immunogen comprises an effective dose of the peptide or protein of claim 20, in admixture with a pharmaceutical excipient.  46. A subunit vaccine formulation, characterized in that the immunogen comprises an effective dose of the peptide or protein of claim 21, in admixture with a pharmaceutical excipient.  47. A subunit vaccine formulation, characterized in that the immunogen comprises an effective dose of the peptide or protein of claim 22, in admixture with a pharmaceutical excipient.  48. A subunit vaccine formulation, characterized in that the immunogen comprises an effective dose of the peptide or protein of claim 23, in admixture with a pharmaceutical excipient.  49. A subunit vaccine formulation, characterized in that the immunogen comprises an effective dose of the peptide or protein of claim 24 in admixture with a pharmaceutical excipient.  50. Recombinant DNA (deoxyribonucleic acid) vector, characterized in that it comprises pv-envl.  51. A unicellular organ, characterized in that it comprises the recombinant DNA vector of claim 50.  52. A bacterium characterized in that it comprises the recombinant DNA vector of claim 50.  53. A bacterium according to claim 52, characterized in that it is or comprises Escherichia coli deposited with NRLL and which has received the accession number B18003 or is a mutant, recombinant or derived from this bacterium obtained by genetic engineering.  54. Recombinant DNA vector characterized in that it comprises pv-env2.  55. Unicellular organism characterized in that it contains the recombinant DNA vector of claim 54.  56. A bacterium containing the recombinant DNA vector of claim 54.  57. A bacterium according to claim 56, characterized in that it is or comprises Escherichia coli deposited with NRLL and which has been assigned the accession number B18004, or a mutant, recombinant or genetically engineered derivative.  58. Recombinant DNA vector characterized in that it comprises pv-env5.  59. A unicellular organ characterized in that it comprises the recombinant DNA vector of claim 58.  60. Bacterium characterized in that it comprises the vector of Recombinant DNA of claim 58.  The bacterium of claim 60, characterized in that it is or comprises Escherichia coli deposited at NRLL and which has been assigned the accession number B-18005 or a mutant, recombinant or derived from this genetically engineered bacterium.