JPH05503629A - Non-replicating recombinant retroviral particles used as antiviral agents and immunogens - Google Patents

Abstract

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

Description

[Detailed description of the invention] Non-replicating recombinant retroviral particles used as antiviral agents and immunogens l. Technical field The present invention relates to recombinantly produced retroviral particles, IL-vitro system that can be used to generate human immunodeficiency virus (HU) Human immunodeficiency virus (HIV) As an antiviral agent and immunogen for the prevention and treatment of toretrovirus. Regarding using this. The recombinant HIV particles of the present invention can be properly processed. It incorporates the synthesized HIV core and envelope proteins, and the morphology It is biologically and immunologically very similar to natural HIV. However, the present invention Recombinant HrV particles contain all elements of the HIV genome necessary for viral replication. It is non-infectious because it does not contain any

2. Conventional technology Regarding human retroviruses, so far, leukemia virus and AIDS or AI Two types of DS-associated viruses have been identified. Main targets of human retroviruses The targets are T lymphocytes and cells of the central nervous system. All human retroviruses are closely related It is transmitted through close contact, blood contamination, and intrauterine infection or breastfeeding after birth. All of Human retroviruses originated in Africa, probably in African green monkeys or related It is thought that humans were infected through interspecies transmission. First discovered Human T-cell leukemia virus type r (HTLV-I), which is a retrovirus, and human HTLV-n (HTLV-n) is a type of T-cell leukemia type 5 virus that consists of T4 cells and some T8 cells. have a tropism for T. Associated with cellular leukemia and lymphoma. Human immunodeficiency virus (HIV) ) is called. Other groups of human retroviruses are detailed below. child There are two major differences between these two types of human retroviruses: 1) Is there substantial genomic diversity among various HIV isolates, whereas HTLV -I and HTLV-I ['s genomes are stable: and (2) HrV is HTLV It entered humans much more recently than HTLV-I or HTLV-II.

21. Human immunodeficiency virus and AIDS Human immunodeficiency virus (HIV) A cystic retrovirus that causes acquired immunodeficiency syndrome (AIDS) . Two types of HrV have been identified so far. Previously, lymphadenopathy-related viruses (LAV) and human T-cell leukemia virus concave (HTLV-I). The prototype virus I-(I”v--■) was reported around the world. It is responsible for most of the cases of AIDS. Another retrovirus HrV-2, which was first isolated from an AIDS patient in West Africa, Pathologically related to V-1. At the genetic level, HIV-2 infects monkeys. It is more closely related to the immunodeficiency virus (SIV), a retrovirus that ing.

By May 31, 1989, more than 97,000 cases of AI had been reported in the United States. DS has been reported, and more than half of them have already died. In the United States As many as 3 million people are asymptomatic) T[V carriers and can transmit the virus. There is. By 1991, there will be 270,000 cases of AIDS in the United States. It is expected that (U, S, Public Health 5 service, 198 6. Public Health Rep, 101:341). AIDS fatality The rate is extremely high, exceeding 80% within 3 years of diagnosis and perhaps 100% in the long term. reach.

It is estimated that there are currently 5 to 10 million people infected with AIDS worldwide.

Especially when there are tens of thousands of people infected with HrV, there are hundreds of thousands of deaths. The statistics from the continent of Africa, where the majority of infections are transmitted between the sexes, are dire. . To date, there is no cure for AIDS and no effective vaccine against HIV infection. There is no sound.

2.2. Pathology of HIV infection HrV is a member of the non-transforming, cytopathic lentivirus genus of retroviruses. Ru. HIV typically causes severe immunodeficiency or neurodestructive disease, or both. It causes a characteristic fatal disease. The main cause of HTVT-induced immunosuppression is Expresses the CD4 molecule, which acts as a high-affinity cell surface receptor for viruses helper/inducer subsets of T lymphocytes (T4 or CD4” cells) that This is due to a lack of resources. T4 lymphocytes induce almost all immune functions in the body are directly or indirectly involved in This results in increased sensitivity.

In addition to T4 lymphocytes, other cells expressing the CD4 molecule, especially monocytes-macro Phages and certain neurons and glial cells in the brain are also targets of HIV infection. Ru. HrV infection also causes polyclonal activation, immunoglobulinosis, circulating This can lead to severe cellular abnormalities, including increased levels of immune complexes and autoantibodies.

A decrease in the number of functional natural killer (NK) cells is also seen in AIDS patients.

Infection of CD4'' cells begins with a combination of CD4 molecules and the major HrV en-rope glycoprotein. It begins with an interaction with gp120, a pyrion protein, and then a pyrion protein. naturalization and exogenous release of genomic RNA by virus-encoded reverse transcriptase. Transcription of the resulting proviral DNA into host cell chromosome D r'J Incorporation into A occurs. Furthermore, it seems that the proviral DNA that was not taken in It accumulates in large amounts in infected cells and is probably a major factor in HIV cell degeneration ( Shaw et al., 1984.5science 226:1165).

During replication, the mR,NA transcripts of the incorporated proviral DNA or the HIV tag translated into protein. This protein is then processed into the HIV genome. It is composed together with nom RNA. Mature pillions are released from the surface of infected T lymphocytes. It also buds internally with macrophages, takes up host cell membrane lipids, and becomes a pilin. Form an on-envelope.

HIV remains latent for a while after infection, but once the virus becomes active and replicates, it becomes invisible. The CD4” cells of the normal host are killed. The mechanism is unknown or unknown.

Some mechanisms have been proposed (e.g. large numbers of unincorporated will Accumulation of viral DNA in infected cells: when a large amount of virus buds from the cell surface Increased cell membrane permeability, induces terminal differentiation of HIV-infected T4 cells and shortens their lifespan. ). Both the CD4 molecule and the viral envelope There is growing evidence that HIV plays a role in cytopathic effects in infected cells. It's coming. A prominent feature in the cytopathology of HrV infection is gp 120/g Multinucleation syncytia thought to be induced by p41 envelope protein This is the formation of um. In contrast, HIV-infected macrophages have long-term HIV can continue to be produced without exhibiting cytopathic effects.

Monocytes and macrophages play major roles in the pathogenesis of HIV infection The evidence is convincing. In addition to swallowing viruses through phagocytosis, Some monocyte-macrophage subsets express the CD4 surface antigen and therefore H It can bind to the rV enherobe. Monocytes-macrophages are infected in the brain the main cell type involved in the formation of neuropsychiatric symptoms of HIV infection are doing. Furthermore, infected patients often have a functional defect in monocytes-macrophages. be noticed. This deficiency may contribute to the characteristic opportunistic infections in AIDS patients. Maybe.

It is important that HIV can survive in a dormant state within monocytes-macrophages. Feeling The stained monocytes are infected with HIV, probably due to the low density of CD4 cell surface receptors. It does not show the cytolytic effect it has on T4 cells. Therefore, monocytes HIV reservoir that ultimately transports the virus to the brain, central nervous system, and various organs in the body It can act as a pond. The virus probably crosses the blood/brain barrier while in monocytes. However, monocytes influence the release of monokines, enzymes, and leukocyte chemotactic factors to leading to destruction or damage of neurons and inflammation of brain tissue (Ho et at, , 1987. N, EnglJ, Med, 31t:278; Fauci, )988.5science 239:617). directly attributable to HIV infection Of the various neurological syndromes, the most common is subacute encephalitis or AIDS dimentia (nearly 90% of AIDS patients), and its clinical symptoms are symptomatic. Including mentia, psychomotor inhibition, and behavioral changes.

2.3. Diversity of HIV morphology and genome The ultrastructure of HIV can be determined using immunological methods and This was elucidated by the use of electron microscopy and electron microscopy analysis techniques (Gelderblom et al. ,, +988. Micron and Microscopia 19:4 1; Gelderblom et al., 1987.　Virology 156:171).

HIV pillions are spherical particles with a diameter of approximately 100 to 120 nm, and p24 ga The electron-dense tubular core consists of g protein, and the submembrane core consists of gag p17. trix, and the env proteins gp120 and gp interspersed between the lipid bilayer membranes. 41 envelopes. HrV genomic RNA is a reverse transcriptase molecule (R enzyme that catalyzes the transcription of NA into proviral DNA) and core protein. It is present within the core as part of a complex containing liponucleoprotein (PNP).

Envelope protein derived from proteolysis from precursor gl) 160 gp120 and gp41 are present in a non-cooperatively associated complex embedded in membranes. Exists as a body. These envelope protein complexes have a maximum width of approximately 14 nm. , observed as a knob with a height of 99-1 On, T=7 repo (laev o) They appear to be arranged in an icosahedral shape that fosters symmetry. This protrusion is gp+2 0, is essential for the efficient generation of this or that tovirion, and in vitro It is loosely bound to the tro lance membrane gp41 anchor. envelope A considerable amount of gp120 is naturally shed from the virus surface, and this phenomenon may be responsible for HI. It may have an effect on V pathogenicity. Virus particle maturation is a budding process It occurs both during and immediately after.

After budding from the surface of infected cells, the HTV core protein is an HIV-encoded protein. It is cleaved from the precursor by rotease to become the mature structural protein; Organize so that they form a core structure.

The HIV genome contains three genes that encode the main structural components of pillions: Env (encodes envelope protein), gag (core protein) pol (reverse transcriptase, protease, and endonuclease), and pol (reverse transcriptase, protease, and endonuclease) coats enzyme). These three genes are LTR (longt sandwiched on both sides by nucleotide structures called erminal repeats. It is rare. LTR contains sequences that play a role in controlling viral gene expression . However, unlike other retroviruses, the HIV genome is small. Both contain six additional genes, three of which are known to have regulatory functions. Ru. It is believed that the expression of these regulatory genes influences HIV pathogenesis. t The at gene is a protein that functions as a potent trans-acting factor for HIV gene expression. encodes protein and therefore plays an important role in amplifying viral replication. rev The gene product controls splicing and transport of HrV mRNA. With this In contrast, the nef gene downregulates viral expression. The v1f gene is pi It may not be absolutely necessary for lyon formation, but it may be a real danger associated with infectious activation. In addition, such traditional fly-mouthing affects virus transmission. Vpr remains The gene encodes an immunogenic protein of unknown function. Finally, the recently announced V The pu oven reading frame is involved in the control of viral maturation and cytopathic effects. encodes the protein that provides the protein.

Many different HrV isolates have been obtained and their nucleotide sequences have been determined. , the surprising diversity of the genome in the env gene has been revealed. selfish aspect Regions of the env gene characterized by differences are shared with common domains among different isolates. scattered throughout. All HIV isolates bind to the CD4 cell surface receptor molecule. So, it's probably one of these common regions or the CD4 binding domain. Related is different HIV-1 variants (some of which are antigenically different) or isolated from individual AIDS patients during the course of infection. Isolates are They differ in their tropism for cell types. In this regard, certain isolates are CD4 y prefer to replicate in either T cells or brain-derived macrophages Apparently, this is a clinical manifestation of HIV infection due to selective pathogenic mechanisms. This suggests different results.

2.4 Vaccine prospects The traditional approach to producing viral vaccines is to use live, attenuated forms or non-viral vaccines. Whole pillions are used in the form of activated preparations. Such an approach has been used to treat smallpox, E. It is used effectively against many diseases such as measles, mumps, and rubella. came. However, regarding the effectiveness of using this approach for HIV vaccines, has been questioned. Reversions and some inadequacies are originally healthy. Since the entire genome of Retrolysul is introduced into the body of a healthy individual, AIDS There is a theoretical risk of introducing diseases such as In the end, the AIDS Most of the development efforts have focused on developing vaccines in the form of subunit or virus-borne vaccines. have been directed towards recombinant methods approaches.

Most studies of HIV target antigens have been limited to envelope glycoproteins. , and there are fewer studies on core antigens.

Knowledge of the immunogenicity of envelope glycoprotein complexes (i.e., soluble gp 120 or gp160, gl)120-gp41 multimeric complex), There is little, if any, knowledge about the envelope-core antigen complex. That's it. Based on some evidence, envelope glycoproteins in specific structures It has been argued that the appearance of both the protein complex and the core antigen is important.

First of all, research on hepatitis B surface antigens has shown that as part of the particle structure, The appearance of this antigen suggests that it is more effective than soluble antigen (Cabra l et al., 1978. (J, Gen, Virol, 38:339) . Second, certain epitopes, such as group-specific neutralizing epitopes of the adenovirus hexon, The immunogenic properties of pitopes may depend on their conformation.

Third, the core antigen of HIV is relatively common in many isolates, making it a useful immunogen. Broad reactivity against different HIV-1 isolates by including core antigens It may also be possible to elicit an immune response. Therefore, in combination with traditional approaches, A vaccine that combines the advantages of both synthetic approaches, i.e. immunization of native pillions. whether it retains its pathogenic properties, the infectivity of all virus preparations, or other undesirable Are you interested in designing and evaluating recombinant vaccines that have no specific properties? It became important.

This vaccine can be used not only prophylactically, but also for post-infection immunotherapy. . For example, the rabies vaccine is currently not available for use in people who may have been infected with the rabies virus. being administered. Because there is a long incubation period between infection and the onset of the disease, HI Is giving immunotherapy to V-infected people valuable in preventing AIDS? has been proposed. (Salk, 1987. Nature 327:473 -476).

3 Disclosure of the invention The present invention relates to non-replicating recombinant retrovirus particles, non-replicating recombinant retrovirus particles, and non-replicating recombinant retrovirus particles. Vaccine preparations containing particles, methods for producing non-replicating recombinant retrovirus particles, and Concerning the use of non-replicating recombinant retroviral particles as viral agents. present invention The recombinant retrovirus particles have an immune system similar to that of natural retrovirus pillions Retrovirus core and envelope structured into structures with physical and morphological characteristics Contains protein. Recombinant retrovirus particles of the present invention and natural retrovirus particles The main structural difference from the virus particle is that the former contains a complete retroviral genome. That's a good thing. Among other things, some of the factors necessary for retrovirus infectivity and replication. Lacking a retroviral genome capable of directing the expression of gene products Therefore, the recombinant retrovirus particles of the present invention are completely non-infectious and cannot be reproduced. I can't do that. However, recombinant retrovirus particles are infectious retrovirus particles. Because it is structurally similar to its offspring, it is highly immunogenic and It is difficult to elicit a protective immune response against a particular retrovirus; It is also effective in blocking virus infection.

The method for producing non-replicating recombinant retrovirus particles of the present invention by the applicant is Directs the maturation and correct organization of virus core and envelope structural proteins in mammalian host cells capable of supporting their association into budded particles. , co-expressing the above-mentioned retrovirus core and envelope structural proteins. including. Such retrovirus core and envelope in mammalian host cells. To introduce a nucleotide sequence encoding a structural protein, for example, Infection with virus vectors and transfection with DNA vectors can be carried out using established techniques. In addition, retrovirus core data Retroviral proteases are used to ensure correct processing of proteins. It is also necessary to introduce the nucleotide sequence encoding the enzyme into the mammalian host cell. I think so.

More particularly, the present invention provides non-replicating recombinant HrV particles, human immunodeficiency virus vaccine against the virus, a method for producing non-replicating recombinant HrV particles, and methods for suppressing HIV transmission. and the use of non-replicating recombinant HIV particles to treat individuals infected with HIV. related. In certain embodiments described herein by way of example, native HIV-1 and mammals that direct the production of HIV-1 type particles with similar immunological and morphological characteristics. human immunodeficiency virus gag, protease and envelope in mammalian host cells. Recombinant vaccinia virus was used as a vector to introduce the Rope gene. There is. These recombinant HIV-1 particles block the infectivity of live HIV in vitro. It is highly immunogenic in vivo.

4 Brief explanation of the drawing Figure 1 shows the expression in B5C-40 cells infected with recombinant vaccinia virus. Figure 2 shows radioimmunoprecipitation analysis of HIV-1 proteins. B5C-40 cells The layers were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS). The cells were grown confluently in (DMEM). Cells were incubated with v-env5 (lanes A and B). ), v-env5+v-gag2 (lanes C and D), v-gag2 (lanes Either lanes E and F) or v-NY parent virus (lanes G and H) were added to each column. 10 PFU of virus/cell. At 12 hours post-infection, cells were infected with [3 5S]-methionine and r35S]-cysteine (100 uCi/ml) at 4 hours. radioactively labeled. Collect the medium, wash the cells with PBS, harvest and RIP Buffer (1% NP40.0.5% deoxocholic acid in PBS, 0.1 %5DS). Cell lysate after nucleus removal (lanes A, C, E and and G), and in parallel using culture medium (lanes B, D, F and H). HIV-1 protein was assayed by RIP containing natural anti-HrV-1 serum. (see sections 6, 1 and 2 below), followed by 11.5% acrylamide tomato. fractionation by 5DS-PAGE in Rix. The radiolabeled protein is Observed by autoradiography. Molecular weight markers are expressed in kilodaltons (kD). did.

Figure 2 shows HIV envelope proteins synthesized by infected B5C-40 cells. The cell surface compartmentalization of the cell surface is shown. MOT of +oPFU/cell for each virus. Infect cells with v-env5 or simultaneously infect with v-env5 and v-gag2. and parallel infection with the parental v-NY virus. 16 hours after infection, medium was aspirated, the cells were washed and 0.5 m C1 was radiolabeled with 125I (Haffar et al., 1987 ．． Mo1. Ce11. Biol, 7:1508). followed by postnuclear cell lysis Figure 1 and section 6.1.2 below. As described in the section, R[' and 5DS-PAGEI:: thus assayed HIv protein.

Figure 3 shows isolation of recombinant HIV particles containing HIV-1 gag and env proteins. shows. (1) Autoradiogram: B5C-4 infected as described in Figure 2 0 cells were treated with [35S comethionine and [35S cocysteine] 5 hours after infection. The cells were radiolabeled for 10 hours using 60 μCi/ml. Cultures from each infection condition Cells were collected by collecting the solution (14 ml) and centrifuging at 600xg for 10 minutes. Clarification was performed. 2 ml of the resulting supernatant was collected and assayed for starting material (TS). It was used for. The remaining 12 ml from each sample was divided into 12 ml in a 5W55Ti rotor. 0. Granular pellets and post-grain supernatant by treatment with oooxg for 3 h. It was fractionated into Mi (S). Rinse the pellet and resuspend in PBS with 15% solution. Placed on 2 ml of sugar cushion. 120.000xg in 3W55Ti rotor The particulate material was again sedimented by ultracentrifugation for 1.5 hours at . grains obtained The shaped pellet (P) was resuspended in RIP buffer. Section 6 below P fraction (lanes B, E and H) was purified by RIP as described in Section 2. HIV protein was assayed, and in parallel, 2 ml of S fraction (total volume 12 ml) was assayed. (lanes C, F and I), and 2 ml of TS material (lanes A, D and and G) were also assayed. (2) Graph, culture supernatant of superinfected B5C-40 The granular pellets obtained from the first superreduction of 60%) in a 5W55Ti rotor at 120,000xg for 1.5 hours It was allowed to settle for a while. A 200 μm gradient fraction was collected from the bottom of the gradient. recovered material section 6.1.2 below. As stated in section Therefore, gagp24 was assayed. EIA peak fractions for each collected fraction Detection of p24 in ng (black circles) and correlated with soyosaccharide concentration (white circles).

The fractions consisting of the top of the gradient (not shown) were determined by Western blot analysis. Well, it didn't contain any p24.

Figure 4 shows the constructed recombinant HIV- Analysis of one particle is shown. V-env5 and v-gag2 (MO for each virus Complete 5C-40 cells co-infected with I = lOPFU/cell) and culture supernatant. In parallel with the granular pellets (Fig. 3) that had settled from The cells were fixed for 20 minutes. The samples were then washed and treated with 08% false serum solution in PBS. Blocked with rubumin, 01% Seratin, and 5% normal goat serum. Various of the above MAbs as ascitic fluid (1:2000 in Brono King Vasofer) in a sample of 110-4 or 41-1 (see section 6, 1.2 below) (see).

After 3.5 hours, samples were washed with PBS and incubated with gold-conjugated goat anti-mouse rgG. and prepared for EM analysis according to the method described below in Section 613. Manufactured.

Figure 5 shows 6.2.4. Todd plot hybridization assay described later in Figure 2 shows the nucleic acid content of recombinant HIV-1 particles as measured by A. Panel A: gag-specific probe. Panel B: enV-specific probe. Recombinant H rV-1 particle and inactivated HIV pyrion concentrations were measured as ng p24 equivalents. (Inactivated virus: lane 1, 2600 ng p24; recombinant H V particles: Lane 1.300ng p24), panel C: recombinant vaccinia virus v-g gag-pot gene (25 8-33]7) and env gene (5671-8572) are shown by lines. arrow The mark indicates the RNA backing sequence (300- 319) indicating the position of (Lever et at,, 1989. J, Vir ol.

63:4085-4087).

Figure 6 is a schematic diagram showing the construction of plasmid pv-G2E5. Figure 7 is a schematic diagram showing the construction of plasmid pv-G2E5. A Western plot of cells infected with recombinant vaccinia virus is shown. B5C-4( [One cell was infected with a Mol of 5 PFU/cell, collected 24 hours after infection, and PAGE I did it. Electrophoresis of infected cell lysates was performed in a 7-15% acrylamide gel. and electrotransferred to nitrocellulose. Immunoplot with HIV + human serum ( Trima r) and then peroxidase conjugated goat anti-human I It was reacted with gG. Visualize the plot using 2-chloro-naphthol as substrate It became. Gel lanes were arranged as shown in the figure.

Figure 8 shows recombinant products produced by B5C-40 cells infected with V-02E5. Figure 9 shows radioimmunoprecipitation analysis of HIV-1 particles. This will be explained later in the section l. A schematic representation of the Blasmitohector construct is shown. A: CmHIVdelXmn (1 133-a and CmHIVde IKpnAvr (Gag2TRE) (+1 60-al), B:CmHiGag2Rre (1158-al') and Cm vGag2Rre (1159-al), C:CmHiEnv5 (l 1.0 4-b 1) and CmE (iTgfbEnv5 (11,13-al), D: BsMkTat (1103-1), BsMtRev (1102-2), CmH iRev (1132-c]) and CmvRev (1152-a I).

Figure 1O shows recombinant HIV-1 produced in transfected CHO cells. Showing the immunoreactivity of the particles. Recombinant HIV particles were extracted from the culture medium of 3010-C6 cells. Collect, concentrate by high speed centrifugation, and bundle in a 15-60% sucrose gradient. It was fractionated by Top: protein yield of gradient fractions using Gag antigen ETA I was analyzed and sucrose density was analyzed using a refractometer. Bottom selected fraction (first A small amount obtained from 7-15% gradient selective fraction polyacrylic This was electrophoresed on an amide gel and electrotransferred to a nitrocellulose filter. is human A r D S, =.

The search was performed using human serum and +25-1 labeled protein A. Unfractionated particles and isolated A small amount of HIV virus was also analyzed.

Figure 11 shows plasmids encoding HIV-1 env, tat and reV genes. HrV-specific expression in HeLa cells transfected with the vector. Analysis of foreign antigens is shown. HeLa cells were treated with CmHiTgfbEnv5 (each lane ) and tat and rev plasmids and tejiBs” as shown on each lane. From “Bluescribe plus” vector without inserted coding sequence A control plasmid) was co-transfected. sample Zinc was added to the medium 24 hours before harvesting, and this is indicated by "+Zn" in the figure. Whole cell regeneration Zeite was analyzed by electrophoresis on a 10% polyacrylamide gel and Nitrocell 125-■ labeled monoclonal antibody 1 ．． Searched on 10-4. B5C- infected with vaccinia virus v-env5 A small amount of 40 cells was also analyzed.

Figure 12 shows Western profiling of cells infected with recombinant vaccinia virus. B 5C-40 cells were infected with mol of 10 pfu/cell and harvested 23 hours after infection. PAGE was performed. Electrophoresis of infected cell lysate on 8.5% acrylimide gel and electrotransferred to nitrocellulose. Immunoplot with HIV + human serum ( Trimar) and then peroxidase-conjugated goat-anti-human rgG I reacted. Visualize the plot using 2-chloro-naphthol as substrate Ta. Gel lanes were arranged as shown in the figure.

Figure 13 shows the analysis of infectivity of T-lymphoblastoid cells using recombinant HIV-1 particles. shows. T-IJ lymphoblastoid cells (CEM) were assembled as described below in Section 71. Recombinant HrV-1 particles (corresponding to 3 ng of p24 gag) or H[V virus (corresponding to 5 pg p24 gag). infection After 5 days, cell samples were collected from each well and cultured as described below in section 71. Intracellular HIV antigen was analyzed by indirect immunofluorescence. Evans Blue dyed The distribution of cells was examined using dye (red staining). Panel A: Recombinant IIHIV-1 CEM cells incubated with particles. Panel B: Together with the HIV virus CEM cells incubated with: Q panel C showing positive fluorescence image: Synsotium of CEM cells incubated with HIV virus is shown. .

Figure 14 shows (A) recombinant HIV-1 particles, and (B)':j') Laren inactivated Serum samples collected from New Zealand rabbits immunized with HIV-1 indicates the relative antibody titer determined by ELISA. For assay details see 1. Figure 15, described below in section 4.1, shows the humoral immune response of the immunized animals. new Recombinantly produced HIV-1 particles (R#238 and R#241) from white rabbits and inactivated with HIV-1 pyrions (R#239 and R#243). − After the next immunization, serum samples were collected at various intervals and whole virus (panel) was disrupted. A and B) or purified gp1-20 (panels C and D) using ELISA : Therefore, rHrV-1 specific antibodies were assayed. The data shown (ordinates) are Endpoint titers of HIV-1 specific antibodies calculated as twice the pre-epidemic serum titer are represented. Yokoza Marks represent the number of replicates after primary immunization at the time the serum samples were collected. Arrows are quadratic 4 weeks R#241 and 243, 5th week R#238 and 239) and tertiary (18th week R#238 and 239) R#241, week 33 R#238) Indicates the timing of immunization.

Figure 16 shows neutralization of HIV-1 infectivity of CEM cells by rabbit serum. exemption Selected serum samples from infected rabbits (Panel A R#241, Panel B R#239 and R#243) to assay HTV-1-specific neutralizing activity. Ta. Homologous virus (BRU isolate) was incubated with appropriate serum at 37°C before addition to cells. Breincubate for 45 minutes at °C. After 1 hour at 37°C, the virus and Serum was removed from the cells and replaced with an appropriate dilution of serum in the medium. A cell? To measure the decrease in p24'' protein released from We decided to neutralize it. The reported neutralization titer (ordinate) is the p24 level in the medium. This was against a 75% decrease in The abscissa is the value mentioned in the explanation of FIG.

Figure 17 shows the interaction of antibodies with individual viral proteins measured by Western blot. Shows reactivity. Details of the experimental method and examination of the results will be discussed later in +4.2.4. It is described in the section.

Figure 18 shows recombinant HrV-1 particles and subsequent incubation with HIV-specific antibodies. Con of HeLa cells transfected with the activated CD4 gene. Figure 3 shows a focal racer scanning micrograph. Details of the experimental method will be explained later. Please refer to Section 15.1.

5 Detailed description of the invention The present invention relates to a live retrovirus pilin in its immunological, structural, and morphological properties. Concerning non-replicating recombinant retroviral particles that are very similar to on. Inventor The method is applicable to all human retroviruses (e.g. HTLV-1, HTLV-n, H IV-1, 8IV-2) can be applied to the construction of recombinant particles similar to .

Certain aspects of the invention relate to recombinant HIV particles. Recombinant HIV particles are As with natural HIV, the correct protein that retains immunoreactivity to anti-HrV serum Contains processed and assembled HIV core and envelope proteins.

However, recombinant HrV particles do not contain the HIV genome and are therefore non-replicating. It is true. The method of the present invention provides for g120/gp41 envelope proteins on the surface thereof. Novel inv for producing recombinant HIV-1 particles with protein complexes This is illustrated by an example using the itro system. The invention encompasses numerous aspects. Everything that falls within the scope of the appended claims is not specifically stated herein or Those skilled in the art will understand that the invention falls within the scope of the present invention even if it is not illustrated.

Several features make the present invention a novel approach to AIDS vaccines. It is distinguished by First, recombinant HIV particles have both morphological and antigenic properties. It closely resembles the true HTV pillion. When used as an immunogen, these The particles provide antigen in a manner similar to the antigen donation that occurs during HIV infection. Therefore, it is highly relevant to natural infection and does not elicit a protective immune response. Ru. This feature of the invention applies to any recombinant subunit vaccine described to date. This was not achieved even by the government. Second, the present invention is based on recombinant DNA techniques. approach incorporates antigens from diverse isolates of HIV-1 and HIV-2. This flexibility is essential for an effective vaccine against A[)S. This may result in a cross-reactive immune response that is thought to be of interest. recombinant dna hand The law also targets harmful epitopes that contribute to making the virus more infectious and pathogenic. It can be used to delete or modify a group. Third, here we describe The recombinant HIV particles are not infectious and do not contain the complete HIV genome. subordinate Therefore, immunization with these particles results in an inactivated or attenuated whole virus vaccine. There is no risk of possible infection associated. These and other features of the invention This will be further explained in the following sections.

The recombinant HIV particles of the invention also boost the immune response against the virus. to prevent the progression of AIDS in individuals already infected with HrV. , can also be used as a specific immunostimulant. This aspect of the invention Enhance or maintain immunoprotective factors already introduced into the active individual The purpose is to A similar approach would be to use a dead, non-enveloped HI It has been evaluated by Dr. Jonas 5alk using the V formulation.

Recombination of the present invention! Other intended uses of RHIV particles include preventing HIV infection. Use as an antiviral agent against HIV core and envelope protein antigens. Used to raise monoclonal antibodies to produce anti-idiotypic antibodies use for, and HIVz encapsulation. ) including its use to clarify processes. The recombinant HrV-1 particles of the present invention are anti-inflammatory. (See Sections 12 and 13 below) which exhibit viral activity, and rabbits immunized with the particles. elicits HIV-specific humoral and cellular immune responses in both short-legged monkeys and short-legged monkeys; (See items 14 and +6, respectively).

Applicant's method of producing recombinant HrV particles is based on HIV en~.-encoding and and co-expressing gag-encoded structural proteins in mammalian cells. nothing. The selected cultured host cells synthesize and correctly process HIV proteins. It must be possible to Introduction of env and gag genes into host cells Live viral vectors such as vaccinia virus and retroviral vectors This method includes infection by vectors, and transfection using DNA vectors. This can be accomplished using a variety of industry-established methods. HI in host cells After successful expression of the V protein, the culture medium is removed by standard methods in the art. It is possible to isolate 817 particles.

In certain embodiments detailed below and described in the Examples in Section 6 below. In this case, recombinant H1V particles are derived from two types of recombinant vaccinia viruses (one is infected with HIV - one with a complete gag gene and the other with a complete env gene) at the same time. Produced in infected African Mitrician kidney II (BSC-40) cells. Ta.

This double infection results in recombinant HIV- One particle sprouted. Biochemical analysis reveals that these particles are mature and immunoreactive. It became clear that gag and env proteins were incorporated. with electronic microscope Apparently, the morphology of the recombinant particles is virtually the same as live HIV.

In a related embodiment, recombinant retroviruses of the invention using viral vectors Another system for producing particles of HIV-1 is shown in the example, and here Breastfeeding using one recombinant vaccinia virus containing both v and gag genes. The mammalian cells are then transfected with recombinant H.I.V. -1 particle is produced (see Section 7 below).

In other embodiments, the DNA encoding the retroviral structural proteins is Recombinant retroviruses are produced using a system containing transfected mammalian cells. Particles can be manufactured.

In an example of this aspect, described in further detail in Section 8 below, HrV-I gag and Using two plasmid vectors each encoding the HrV-1 env gene. The CHO cells were then transfected with recombinant H Directs the synthesis of HIV-1 gag and env antigens organized into IV-1 particles . Other methods related to this aspect include creating complex plasmids containing multiple HIV genes. transfection with vectors to promote HIV gene expression. Use of constitutive and regulatable enhancer/promoter elements that allow HrV control Expression of proteins, expression of HIV structural genes, use of different cell lines, and modification of H ■Includes or is limited to the use of plasmid vectors encoding v proteins. It's not something you can do.

Regarding other methods for expressing HIV structural proteins and for producing recombinant HrV particles. For example, 8, 3. , 9 and 10.

Using the system of the present invention, many options are available to control the properties of the resulting particles. It is Noh. These selections are made at both stages of virus construction and infection. can.

5.1.1 Production of recombinant DNA and viral vectors such as vaccinia virus Recombinant DNA vectors and viral vectors are disclosed in pending U.S. Patent Application No. 779. ．． No. 909 (September 25, 1985, issue 8): No. 842.984 (1986 No. 905.217 (filed on September 9, 1986), can be constructed according to the methods outlined in , each of which is incorporated herein by reference in its entirety. use

In a particular embodiment of this study, a recombinant vaccine that also contains the HIV env and gag sequences Construct a senior virus and use it as a vector. In short, vaccinia The former core and envelope protein coat sequences are under the transcriptional control of the promoter. A plasmid vector containing the sequence was constructed and used for in vivo recombination. Integration of the IV gene sequence into the vaccinia virus genome by Ru. Recombinant vaccinia virus as described in the above-mentioned pending patent application. to identify, purify, and demonstrate its ability to direct synthesis of HIV proteins in infected cells. evaluate.

Another embodiment uses a divine combination of classical structural and/or regulatory genes. A recombinant plasmid vector encoding the recombinant H[V particles is formed. Use as a vector for transfection of cells. like this The construction of a representative range of vectors is described below in Section 8 et seq.

The very nature of the individual protein components of the recombinant particles of the present study can be determined using recombinant DNA. During the construction of A vector, recombinant vaxonia virus vector, etc., recombinant DNA technology It can be modified by technique. This method allows the identification of retroviral epitopes present on the particles. It is possible to define the existence and structural composition of For example, lI[V g Variable epitopes of P120 can be involved in the generation of cross-reactive immune responses. Similarly, Various old V gag gene sequences were inserted into recombinant vectors to produce recombinant H[V particles. It is possible to change the immunogenicity of Vector encoding the mutated old gene sequence It can also be useful in the formation of recombinant antiviral, immunogenic and antiviral antibodies. Effects etc. can be improved. Applicant has proposed that such modified core and/or envelope Recombinant retroviral particles that have inserted protein are Wanted ii! intended to be within the range.

5.1.2 Recombinant vectors and recombinant viruses that form recombinant torovirus particles. The nature of the torovirus particles depends on the assembly used in the infection or transfection method. The difference depends not only on the combination of new vectors but also on the combination of vectors used. This choice can be fundamentally varied in all methods of the invention. Tsutsunaka's explanation As a single recombinant vaccinia strain carrying the old V-1 gag gene (v-gag2) When BSC-40 host cells are infected with Applicant has discovered that a large number of particles are formed. But why are these same A recombinant vaccine whose cells contain v-gag2 and the old V-1env gene (v-env5) In the case of co-infection with seniors, the resulting structured particles and expressed this protein on its surface (see section below). 6). Therefore, after infection with v-gag2, particles consisting only of core protein or The addition of v-env5 to the infection system inserts the envelope protein. into large dermal complex particles. Taking the above explanation one step further, the host Cells were transfected with env genes from v-gag2, v-enV5, and H[V-2. Co-infection with a third recombinant vaccinia containing HIV-1 core as well as HIV -1 and former V-2 envelope proteins are formed. It would be expected that Separately, a single protein coats multiple HIV genes. A recombinant vaccinia virus can be used as an infectious vector. less sexy One such vaccinia virus vector, as described in the Example below. Infected host cells also form immunoreactive recombinant HIV-1 particles. .

Similar principles are used to transfect host cells using plasmid vectors. Applications for the formation of recombinant particles using other vector systems, such as for example , host cells are infected with a single plasmid vector encoding H[V env and gag. or HIV env, gag, and vectors encoding Ike's old ■ genes. - can be transfected. Each cell has a different HIV gene or is transduced with multiple plasmid vectors encoding combinations of H[V genes. It can also be effected. Additionally, the transfected cell lines were used to form particles. The system was retriggered with a vector primed to add expression of the Ike's HIV gene. Can be transfected. Using a vector encoding a regulatable promoter, Expression of HIV proteins in transfected host cells can be regulated (e.g. (see section 8.3.2 below). Vectors encoding other HIV genes The H+V structural gene (along with the expression of electrons) was You can express them. Such H[V regulatory and/or auxiliary proteins Expression of protein in the system can be used as a means of altering particle properties and/or their production levels. It can be used as

It is important to note that particles cannot form in the absence of the gag protein. , therefore essential in recombinant vectors) (1) Containing the core protein gene sequence It is necessary. Is HIV protease necessary for core protein composition? , its role in the formation of infectious pillions has been suggested (Peng et al. l,, 1989. Virology 63: 2550). In this way, natural Production of recombinant HIV particles with HtV protease function similar to that of the pilion. It can be advantageous to include it in the structure. Furthermore, H[V-1, HrV-2 or its heel Using gag genes from different isolates, 11% of recombinant vectors can be made. Current As will be apparent to those skilled in the art, the multiple infection method described above and other Similar methods using recombinant vectors can be used to detect a wide range of surface and core antigen characteristics. can form recombinant old V particles with Many different combinations are essentially uncontrolled Limited.

The particular host cell chosen also influences the properties of particles produced by the method of the invention.

Cells have the ability to photoexpress and accurately process mature H[V proteins. should be selected accordingly. H[V envelope proteins gp120 and gp41 is glycosylated and cleaved from the large gp+60 precursor by proteolytic cleavage. cells capable of directing these post-translational processing modifications. desirable. Of course, when protecting recombinant viral vectors, host cells must It must be easy to be infected by new viruses. In a preferred embodiment of the invention , using host cells of human, monkey, or stratozoan origin.

Host cells were grown with recombinant vaccinia virus according to the conditions described in Section 6 below. recombinant plasmid vectors and described in Section 8 et seq. below. can be transfected with a vector. Recombinations as described below in Section 6 et seq. When using a vaccinia vector system, cells are infected with each recombinant vaccinia virus. can be infected at a multiplicity of infection (QIO+) of approximately 10 PFU/cell. However, Those skilled in the field can increase the MOI for one or more recombinant vaccinia. Understand how adding or decreasing can affect the properties of the particles being 1) It will be. This is an important factor in designing multitropic or heterogeneous particles. Dew. For example, H[V-I NHIV-2 envelope resistance on the surface of foreign particles] The desired ratio of can be achieved by infecting with proportional corresponding Mol ratios.

Specific implementations of the invention 1r, detailed in Section 6 below! The recombinant vaccine Using the senior virus V-enV5 and v-gag2, cultured African green Monkey kidney (BSC-40) cells were co-infected. Infected B5C-40 cells were infected with H IV-1 envelope proteins gp120, gp41 and gp160 precursor, array of HIV-1 gag proteins p24, p17, p15, p 55, p45 and p39 were synthesized. Furthermore, at least p24, p17 , gl) +20 and gp41 with polyclonal anti-HrV-11frl serum. , p17, p24, gp+20 and gp41-specific monoclonal antibodies constitute immunoreactive particles. Thin-section electron microscopy and immunometallurgical disorders (immu) 'M of recombinant) 11V-1 particles by nogold labeling) 3m fine structure analysis showed substantial morphological identity to native old V.

In this regard, recombinant barrel V-1 particles are spheres with a diameter of 1100-120n. It is visualized to be a shaped object, and depending on the intercept angle it can be either rod-shaped or spherical. The child contained a dense inner core. Monoclonal against I) 24 and p17, respectively. (by immunoassay and immunoelectron microscopy analysis) confirms the identity of the HIV core structure. H[V-1gp120 and gp41 specific gp12 by immunoelectron IjI microscopic analysis using different monoclonal antibodies. It was shown that 0/gp41 aggregates appear on the surface of recombinant torso v-1 particles.

Immature particles of various morphologies were also visualized and consistent with the course of HIV-1 pillion morphogenesis. I did it. These morphologies were observed from substantially similar analyzes of old V-1 pillions. (The electron ga micrograph shown in Fig. 4 was used by Gelderblom e tal, 1988. Micron and Microscopia 1 9:41 and Ge Derblom et al., 1987. J, Vi (compare with that published in Rol, 156:171). The inventor In conclusion, the M! of these recombinant) 11V-1 particles! The fine structure is The ultrafine structure of viable H [V-1] may be due to the fact that it does not contain enzymes or reverse transcriptase. It's just different.

The recombinant body V particles of the present invention can be used for HIV gag, env and HIV remains in ponds. cells transfected with a recombinant plasmid vector encoding the gene. Can be done. Various specific implementations of aspects of the invention are described below in Section 8.2. to Describe it. In certain demonstrated embodiments, Chinese hamster ovary (CHO) cells are , encodes the HIV-1 gag, env, tat and rev genes Transfect with plasmid vector. Recombinant H+V-1f’ffi child Stable C that expresses and secretes enV and gag proteins processed as Obtain the HO cell line.

Similar results or transfection with different plasmids or combinations of It can be obtained using He1a, BSC-40 and Vero cells (e.g. See section 8.3 below. l, see 8.3.2 and 8.3゜3. ).

5.2. Non-replicating recombinant product containing immunoreactive core and envelope proteins Particle Identification and Isolation Recombinant particles can be isolated using various immunochemical methods and/or electron microscopy. Can be identified by speculum (Elil) visualization. radioimmunoprecipitation (RIP), Capture enzyme immunoassay (EIA), Western blot analysis, etc. Other immunochemical detection methods can be used. Recombinant using these techniques as Specific examples of how manufactured HIV particles can be identified are provided in Section 6 below. Described by. Section 6.1.3 below. Thin-section EM and immunoelectromagnetic Using various 2M techniques, such as microscopy techniques, silk replacement restoration 1. can be determined and characterized. Another 2M technique that can be used is scanning electron microscopy (S EIJ), surface reconstruction electronic button microscopy and immunoclio ultramicrotomy (immunocryoultramicrotomy), all or H1■ It has been proven to be useful in elucidating the narrow beak formation (Gelderbl.

m et at, 1988. Micron and Microscope ia 19° old).

Recombinant particles are isolated from the host culture medium using standard techniques known in the art. can. However, in order to maximize the immunogenicity of recombinant recombinant 1 amplified gp It is important to use a simple separation method that yields a +20 degree of shedding.

5.3. Immunogenicity of non-recombinant recombinant silk particles: The immunogenicity of central recombinant particles is This can be determined by monitoring the immune response of the test animal after immunization. The test animals , mice, rabbits, chimpanzees, and ultimately humans. go There are five routes that can be considered: oral, intramuscular, intramuscular, intravenous, subcutaneous, Including intranasal. There were three immune responses induced by recombinant H[V particle immunosuppressants. (a) Enzyme immunosorbent assay (ELISA) ), immunoplot iff, radioimmunoprecipitation, etc. The reactivity of the obtained immune serum against the authentic HIV antigen using b) In vitro H]■ Ability of immune serum to neutralize infectivity (Robert -Guroff, 1,985. Nature 316:72) and (C ) Protection from HrV infection and/or attenuation of infectious symptoms in immunized animals (F rancis, 1984. Lancet 2: 1276: Gujdus ek, 1985. Lancet 1:55).

In certain embodiments, isolated recombinant HrV-1 particles (Section 6 below) were analyzed for immunogenicity in rabbits (see Section 14 et seq. below). these The analysis results show that the particles are characterized by both the former V envelope and core structural proteins. Because it elicits distinct humoral and cellular immune responses, recombinant HIV-1 It is suggested that the particles are highly immunogenic. In addition, the HfV-1 manufactured by Shokai Rabbits immunized with the particles produced neutralizing antibodies to HIV-1.

In a related embodiment, the immunogenicity of recombinant H+V-1 particles is determined by (see Section 16 et seq. below). More specifically, macaque monkeys are immunized with recombinant HIV-1 particles along with local HIV-1 antigen. sense of immunity Immune responses in cultivated subjects can be determined by various assays, including whole pillion ELISA, g Includes p120 ELISA, focal immunoassay and lymphoproliferative response analysis . As described by the Examples below in Section 16 et seq. Single color of sensitization f5: When used as a source, the HIV-1 particles made by Shodai are I ( IV - elicits specific humoral and cellular immune responses. Recombinant shell V-1 particles is a recombinant cottonsonia virus that coats HIV-1 env and gag antigens. It has been particularly effective when used to immunize animals that have been previously immunized.

5.4. Vaccine prescription A particular embodiment of the invention is that retroviral infections or retroviral infections such as -1[Ds Can trigger an immune response that contributes to protection against virus-associated disease development This is a vaccine prescription. Vaccine formulations are based on major retrovirus cores and By combining envelope proteins, the recombinant protein of the present invention is polyvalent in nature. Prepared retroviral particles are used as immunogens. Related implementation q! , in Mr. Recombinant retrovirus particles can be used in humans already infected with retroviruses. special immunity that can be used to ameliorate disease development associated with retroviruses. A further particular embodiment of the present invention, which can be used as a pre-epidemiological enhancement, is vaccines that can elicit an immune response that helps prevent the spread of AIDS. Including prescription. Such vaccine formulations utilize recombinant Kasei 1a as an immunogen. do.

Traditionally, viral vaccines have been produced from attenuated or inactivated whole pillions. Ta. Mainly used for large-scale virus production (danger, possibility of incomplete activation, etc.) Because of the introduction of the HIV genome into a healthy recipient population, the method also works against old V-1. It was not desirable as a cutin design (Minor, 1989. J. Antimicrobial Chemotherapy 23.5upp, A: 55). Therefore, we are focusing on H[V”7 cutin opening and subunit vaccine candidates. do. Initial interest was in subunits consisting of recombinant gp120 envelope proteins. Was it aimed at knit prescriptions, or is it currently both g[1120 and gp41? To generate neutralizing antibodies (Chahn et al, +986.EM BOJ, 5: 3065: Ho ej al, 1987. J.

Virol, 61: 2024: 5kinner et al, 198 8. J, Viroi, 62: 4195), mediating antibody cell-mediated cytotoxicity. (Tyler et al., 1989. Fifth Inter National Conference on AIDS, Abstract t T, C, 0, 33:521), cytotoxic T, susceptibility to IJ cell death. (2arling et al., 1987, J. Immun. ol, 139:988), and is clearly the target antigen. These consequences As a result, both gp120 and gp41 have been used in the design of HIV-1 vaccines. It was decided that

The association of gp old with the plasma membrane is coupled with its weak non-covalent interaction with gi1120. This makes the production of a completely soluble gp120/gp41 complex impractical.

More importantly, hepatitis B surface antigen (Cabral et all, 1987 ．． J, Ger+, Vial, 38:339) and herpes simplex Rus glycoprotein (Ho et al., 1989. J. Viol. 6 3: 295+), which is the same as the Ike virus antigen that exists as a component of the membrane structure. The membrane-bound gp120/gp41?1i complex is more immunogenic than its soluble counterpart. It seems likely.

As described in the background section of this text, effective older vaccines have been developed. The generation is complicated by several properties of H[V. The present invention solves most of the problems Many, if not all, can be bypassed. In its natural conformation, gag In addition to providing the recombinant and env proteins, the recombinant recombinants of the present invention Designed to retain desired epitopes while deleting or modifying desired epitopes. I can design it. Furthermore, the present invention thereby provides that some of the same-old proteins We present a method that allows different variable epitopes to be inserted into particles for use in vaccine formulations. provide Does the present invention protect against both HIV-1 and HtV-2 infection? We also present a method for making xenogeneic recombinant body V-particles that can be used to formulate vaccines. provide

One of the novel aspects of the vaccination method utilizing the present invention is that these and other It is possible to combine a complete group of such epitopes into one immunogenic particle. difference Furthermore, these epitopes appear to the immune system as they do on natural H[V; induces an immune response that is effective against infection by natural pyrions.

5.4.1. IH[V-1')') Chin Bulletproof immunity to HIV is not completely understood. Not made clear. It is generally believed that both neutralizing antibodies and cell-mediated immunity may be necessary. ing. The reason is that HIV is transmitted in the form of smoke cells or cell-associated forms. . Neutralizing antibodies that recognize many different old epitopes or are identical in HIV-1-infected individuals. 120, a highly conserved region of the envelope protein gl) Includes those that recognize epitopes on variable regions. Similarly, gp41 and p17 Neutralizing antibodies induced against the epitope of (Papsidero et al. , +989. J, Virol, 63:267-272') was identified. There is. Other antibodies detected in previously infected individuals bind to the CD4 cell surface receptor. Contains those specific to the gp120 domain.

Still other antibodies that bind to the hypervariable region of gp120 can be used to isolate HIV-infected cells. (Rusche et al., 1988, Proc, Natl, Acad, Sci, U, S, A, 85: 3 198). Cell-mediated immune responses (cell-mediated immune responses) are also seen in IIV-infected humans, particularly enV, g Identification of cytotoxic T-lymphocytes induced against ag and pot progeny (Walker et at, 1987. Nature 328 :345; N1kon ejal, 1988. Nature336: 484: Riviere et at, 1989. J, Virol, 63:2270-2277).

One practical aspect of the invention is that HIV, which is currently the most widespread form of H[V] -1 virus, and this vaccine is described in section 6 below. Using recombinant shell V-1 particles, such as the recombinant H[V-1 particles described in . set The engineered V-1 particles are structured in a structure that mimics the morphological and antigenic properties of live HIV. It consists of the mature core and envelope proteins of type I viruses. Book Embodiments include vaccine formulations using various recombinant H+V-1 particles. example For example, combining gp120/gp41111 from two or more old V isolates simultaneously inducing the development of protective antibodies against multiple variable region epitopes. It can be useful for The formation of such particles may be due to different former V−+ isolates. env from! ! host with several different recombinant vaccinia viruses, including E. This could be achieved by co-infecting the cells. Immunization with such particles Individuals who have been exposed to the disease may mount an immune response against many different strains of HIV-1. It will be possible.

Similarly, construction of fleshy particles can increase the efficacy of vaccine formulations. This embodiment In some cases, the design is designed to insert epitopes from HIV-1 strains with different tropisms. In. For example, with antigenic determinants common to T4 cell-associated virus strains, a single Recombinant H[V-] particles exhibiting proprietary antigenic determinants in bulb-associated HIV-1 strains are Can be formed using the superinfection method. In particular, such particles can bind host cells to three species. can be formed by co-infection with recombinant vaccines, and IFIs from one strain gag gene, one gene contains the same specific enν gene, and another contains special enν genes with different tropisms.

The most effective vaccine against 1 (+’/-1) is the present vaccine in combination with Ike’s immunogen. The present invention may include the use of recombinantly produced V-1 particles from Ming. In this regard, recombinant H [V-1 particles produced by recombinant gp+60 in non-human primate subjects Humoral and cell-mediated immunity when used as a secondary immunogen after primary immunization appears to be the most effective at eliciting responses.

5, 4.1.2. Old V-2 vaccine and heterologous vaccine Other embodiments of the present invention include: Vaccines against HIV-2 and heterologous vaccines (e.g. H[V-1 and) l[V-2 (which may include vaccines). For use in such heterologous vaccines Recombinant particles containing, for example, old V-1 core protein, HIV-1 and H[V- It can contain two envelope proteins. Apart from this, 2 or It would be desirable to formulate a vaccine consisting of these different recombinant H[V particles.

It protects against many different isolates of both HIV-1 and HIV-2. This is particularly advantageous when designing vaccines.

Vaccines consisting of a single recombinant HIV particle V or a combination of different types are Can be formulated with appropriate adjuvants to enhance the immune response to the antigen . Suitable anionubants include inorganic gels, resolenol, etc. Surfactants such as Chin, Plurionic polyols , polyanions, peptides, oil emulsions and BCG (Calmett e-Guerin rods), Corynebacterium parvum Includes human adjuvants.

A number of methods known in the art can be used to introduce the vaccine formulations described above. For example, skin incision, intravenous injection, subcutaneous injection, intramuscular injection, intranasal administration, oral administration, etc. There is something like that.

6. Example - Formation and isolation of non-replicating recombinant 1 (IV-1 particles) It contains env, gp120 and envelope proteins on its surface. An in vitro system for forming recombinant HIV-1 particles expressing the gp41 antigen was developed. Describe it here. Briefly, BSC-40 cells were infected with a complete envelope of HIV-1. rope gene (v-enz・5) or complete old VI gag and protea Simultaneous immunization with recombinant vaccinia virus containing one of the V-gag genes (v-gag2) dye. HIV-1 protein is expressed in BSC-40 cells and is isolated from the cell membrane. It is composed of budding old V-1 particles. The particles obtained are non-replicable and H[V-1 Reacts with monoclonal antibodies specific for envelope and core proteins, resulting in morphological It is mechanically similar to the old V-t pillion.

African green monkey kidney cells (BSC-40 strain, African green monkey kidney cells derived from BSC-1 cells) Continuous line of Licamitris monkey cells, ATCC number CCL26) with 10% bovine fetal serum. and 100 units/day of penicillin and streptomycin, respectively. Lubetzko's modified Eagle's medium (DMEM, Gibc.

, Grand I5land, NY).

Recombinant vaccinia virus containing HIV-1en~ and gag gene sequences is , pending U.S. Patent Application No. 905.217, (filed September 9, 19814). Manufactured and evaluated as described. Recombinant vaccinia virus v-e nν5) I It has the complete env gene of IV-1. 111% vaccinia virus v- gag2 is a complete combination of old V-1 gag and prt genes α and pot remains (part of standing offspring) to hold.

Vaccinia virus infection in New York City 5.1my (jhe New York City Board of Health 5train) is a Wyeth research Smallpox vaccine (Dry ~・aXLot321501G) was purified from the commercially available t2 preparation. smallpox vaccine Diluted in PBSAtl (see below) and blurred three times in succession on B5C-40 cells. Coo purified. Stocks (hereinafter referred to as V-N Y) was produced on B5C-40 cells and used to construct recombinant viruses. .

6.1.2. Detection of H[V envelope and core antigens using four techniques ・Radioimmunoprecipitation method (Ri P), Capture Enzyme Immunoassay (E[A), Western Proyl. Analysis and Immunization $7. Microscopic examination.

The radioimmunoprecipitation method uses human polyclonal 1-HV-1 serum (Trimar , Inc.) using essentially Haffar et al., 198 8. J, Ce1lBio! , 107:1677.

Briefly, B5C-40 cells were given 10 PFU/cell per virus used. Cells were infected with recombinant or parental vaccinia virus at multiplicity of infection (MOI).

Twelve hours after infection, cells were treated with ["Sl-methionine and [35Sl-instine (10 0 uCi/ml) for 4 hours. Then, collect the medium and PB Wash with S, strain and add RIP buffer (1% NP, 40.05% deoxycohol in PBS). Bacteria were lysed in 1% SDS'). Then post im cell again The culture medium was reacted with polyclonal antiserum for 30 minutes at room temperature. antibody-antigen complex Staphylococcus aureus (Staph A) fixed with 10% glutaraldehyde and room temperature and incubated for 30 minutes. 54aph　A-Antibody-antigen-complex is centrifuged Precipitate with RrT wash buffer (1% NP-40 in PBS, 0, 1% SDS ’) was washed twice. The resulting pellet was subjected to 5DS-PAGE sample buffer (Laem mli, 1970. Nature 227: 680) and solubilize, 11. Immunoprecipitated proteins were fractionated on a 5% polyacrylamide gel and autoradiographed. Visualized with Raffy.

By using capture enzyme immunoassay and Western blotting, Using a monoclonal antibody against p24, Hu et al., +987. Specifically, HlV-1p24 was detected as described in Nature 328:721. I put it out. Used for both capture enzyme immunoassay and Western blotting. The monoclonal antibodies produced were produced using recombinant fusions that define overlapping sequences in the p2-1 protein. was raised against the p24 sequence by immunizing mice with the synthetic peptide. . Monoclonal antibodies 25-2 and 25-3 are disclosed in U.S. Patent Application No. 054.026 ( +987 rod filed on April 30th) and No. 105.761 (filed on October 7th, 1987) Genetic Systems (Washin), N.C., as described in [). generated by Tor).

By using immunoelectron microscopy, M, Ab specific for gp+20 110l10-4 (Tho et al, 1988. AIDS Res, Hum, Retroviruses2:25:in5ley et al ,,+988. J, Virol, 62: 3695) and gp41-specific MAb 41-1 (Gosting et al., 1987. J. Cl1n, crobiot, 25:845) was used to generate recombinant H1 detecting gp120 and gp old envelope proteins on the surface of -1 particles; Furthermore, according to the electronic microscopy techniques described in Section 6.1.3 below, The morphology of recombinant H[V-particles was elucidated.

6.1.3. Electron microscopy Recombinant barrel V-1 particles were separated and prepared for 6M visualization as follows: . Media from 15 hour infected vacuole cultures were pooled. Freeze the culture medium at 6OOXg. The contaminated 8i11 cells were cleared by centrifugation using a top centrifuge. Then the particle fraction 12 It was isolated from the clarified supernatant by ultracentrifugation at 0.000Xg. The obtained supernatant It was collected as post particle material (S). Particle pellet is phosphate buffered saline (PBS) , resuspended at pH 7.4 and added at 120.0 OOX g via a 15% sucrose solution layer. It was reprecipitated by ultracentrifugation. The twice-sedimented material was designated as the particle fraction (P).

The precipitated particle fraction (P) was collected by gently covering the pellet with PBS and then aspirating it several times. washed. The pellets were then fixed in 4% paraformaldehyde for 20 min and PB I washed it again. Similar procedure, B5C40 thin tt'd (10'-10 'cells) were infected with the appropriate recombinant virus. After 15 hours, discard the growth medium and Cell monolayers were washed several times with PBS and then 4% formaldehyde in rose at 22°C. Fixed for 0 minutes.

Thereafter, the fixed cells and fixed particle pellets were washed 5 times in PBS and then washed in PBS. 08% bovine serum albumin (BSA), 0.1% gelatin and northern normal goat blood The cells were blocked for 30 minutes with a solution containing the supernatant (blocking buffer). blocking solution Decant the solution and overlay the cells with the monoclonal antibody (in blocking buffer). ]:2000 dilution) and incubated for 2-3 hours. cells Wash with PBS, then add gold-conjugated goat anti-mouse secondary antibody (coupled to all 15 nm colloids). Combined IgG: Janssen, ++! - Biscataway, Chassis (Piscata growth ay’)) and incubate for another 2 hours at a dilution of 15. , washed with PBS, and fixed with 2% glutaraldehyde. O, ILI force the sample. 1% 4M (hyosmium (Os') in Cacadyfaje buffer) ) and incubated at 22°C for 30 minutes. The sample was then thoroughly added to PBS. Rinse and incubate sequentially in 35%, 50% and 75% ethanol for 3 min. and then dehydrated with 3% uranyl acetate in 70% ethanol for 130 min at 22°C. Intermittent staining. The samples were then heated to 80%, 90%, and 95% maximum at 22°C as described above. Afterwards, the cells were sequentially incubated with 100% ethanol (3 times) and further dehydrated.

The sample was then embedded in methacrylate resin (plastic) as follows. ・Processed for 1 hour with 2:1 ratio of absolute ethanol/plastic, 100% plastic Treated overnight with Rustic. The resin was then polymerized for 2 days at 60'C. Embed the sample Remove the soldered plastic from the plate and cut out a thin piece (1,00 nm). version (f.

m\1ar) was collected on a coated grid.The grid was then replaced with saturated uranyl acetate. / Stain with lead citrate (Millo old g’s) for 10 minutes and wash thoroughly. Purified. After drying, the grid was heated at 60kV using a JEOL 100B transmission electron microscope. Evaluation was performed at a magnification of 1.00.0OOX.

6.2. Non-reproducing recombinant H[V-1 containing mature gag and env proteins particle formation 6.2.1. H expressed in B5C-40 cells infected with recombinant vaccinia virus [Analysis of V-1 protein either v-env5, v-gag2, or both combination, or parental vaccinia virus v-NYI: infected, metabolically radiated Cell lysates and growth media from labeled B5C10 cells were prepared in the section above. 6゜2.1. R [The former V-1 protein was analyzed. As shown in Figure 1, before gp160 env precursor protein and its proteolytic processing production f'ljgp 120 and gp41 were detected in cell lysates of v-env5-infected cells (rare A). Additionally, gp120 was secreted by env5-infected cells (lane B). . The p55 gag precursor synthesized in v-gag2 infected cells is also processed. and mature gag protein 1124, p! 7, p15 and two intermediate fronts giving rise to the precursor species p45 and p39 (lane E) (Gowda et al., 1 989. citation). Interestingly, gag protein was also present in the culture supernatant. (Lane F).

B5C-40 cells co-infected with v-env5, v-ga, and g2 were infected separately. Processed env and g identical to those expressed by the infected cells. ag protein (lane C: compare with lanes A and E).

The mature env and gag proteins formed by these dually infected cells are were similarly localized in the culture supernatant with the same kinetics (lane C: as lanes B and F). (I would like to be compared). In addition, the plasma membrane-associated protein lactoperoxidase Catalyzed iodination induces cell surface release of gp160, gp120 and gp41. The transport to v-en-. It became clear that they were the same (Fig. 2, lane A and infected B5C-10m, respectively). extracellular gp120 and gp41 expressed by to determine whether it is released as a component of the budding particle or as a component of the budding particle. In order to separate the culture supernatant into particle (P) and Bost particle (S) fractions, Section 6゜1.3. ), the H[V protein content of each portion of each portion was determined as 1. ′ It was analyzed by RAP in comparison with f culture supernatant (TS). Figure 3 (1) (lanes A, B and extracellular gp from V-env5 infected cells as shown in C). 20? Mainly detected in the S fraction, this protein is not a constituent of budding particles. I understand. However, cells coinfected with v-env5 and v-gag2 gp120 derived from was detected in the P and S fractions (Figure 3(1), respectively lanes E and F). However, particles ~ association gp! 20 is detectable extracellular gl)+ There was little involvement in the total level of 20.

In contrast to gp120, gp41 was detected only in the P fraction of the L clear from double-infected cells. Once released (Fig. 3 (1), lanes E vs. F), gp41 is attached to the budding particles. It is clear that only one person is meeting. gp160 env’fm precursor or double infected cell It is interesting to note that low levels were detected in the P fraction of the supernatant from the cells. (Figure 3 (1), lanes E versus F).

HIV-1 core protein expressed by infected B5C40 cells was similarly analyzed. Ta. All detectable core proteins - p24, p55, p45, p39 and p17- or v-gag2 infection (Fig. 3 (+,), lanes H and ■ are compared. ), as well as dual infection (Fig. 3(1), compare lanes E and F) on cell culture. I in the P fraction from the clear! I was guessed. The P fraction obtained from double infected cells also Further fractionation was achieved by precipitation on a 15-60% continuous sucrose density gradient to isolate the p24 antigen. Existence as described in Section 6.1.2 above. was evaluated with ErA as in 2a. Figure 3 ( The results shown in 2) indicate that 1124 or a single prominent peak with a 36-40% sucrose fraction indicates that it is separated as a network. These results indicate that the infection was caused by infected B5C40 cells. The expressed gag protein was released as 1htIt of particles of relatively uniform size. Suggests that it will be served. In addition, the P fraction of gp+, 20 and gp old or double infected cells The presence of gp+20/gp41 may be due to particles released from co-infected cells. This suggests that it contains m-coalescence. This conclusion is supported by immunoelectron microscopy analysis (following Section 6, 2.3. Further support is obtained from the results obtained from ).

To directly analyze the morphology of recombinant HIV-1 particles, v-en~·5 and V=g The P fraction separated from the culture supernatant of B5C40 cells co-infected with ag2 was l) Either 1200JAb 110-4) or gp41 (MAb old-1) It was reacted with a crab-specific monoclonal antibody (MAb). Main antigens - anti (*multiple) The combination is further reacted with a secondary antibody-colloidal gold complex and embedded in plastic. See Section 6.1.3 above. Thin section electron microscope as described in ? :1 mirror inspection (E M) was tested. These analyzes revealed that the EM of isolated former V-1 pillion particles i (Gelderblom et al, 1988.1 Jicron an d Microscopia 19:41; Gelderblom et a l,, 1987. Morphologically similar to Virology 156: 171) electron concentration decay (Fig. 4, “a” and “C”) or spherical (Fig. 4, “b”) core. It was revealed that the particles contained approximately 1100-120 nm in diameter. In addition, particles can be When labeled with a gold complex, gp+20 was added to the surface (Figure 4, “a” and “b”). and gp41 (Fig. 4, "C") were shown to be expressed.

Complete 5C-40 cells co-infected with two recombinant vaccinia viruses were A similar analysis was performed using anti-gp120 MAb. 1 positive for gp120 antigen A large number of 100-120n particles were visualized. The majority of these cell-associated particles are Either of two distinct morphologies, one characterized by a diffuse vesicular shape (Fig. 4, “e”, open arrow), while others are identified by eccentrically localized thick bilayer regions. It was estimated that this was the case (Fig. 4, "d", double arrow). The principal liaison shall The different structures of It is assumed that this represents an immature particle with the form of In some cases, a fully formed rod-shaped key The electron micrograph shown in Figure 4, "e" (open arrow) indicates whether the particle contains a capsid structure. It will be seen near the cell surface, as captured by Thin l12! Ill? budding vaccinia particles did not have gp+20 (Fig. 4, “d”, book arrow).

62.4, Nucleic acid content of recombinant HIV-1 particles Recombinant HIV-1r! , child chain The acid content is determined by the “P” RNA probe that reacts with either gag or env sequences. Determined by dontobucott hybridization using . The probe is Promega Riboprobe in the presence of radiolabeled nucleotides Contain gag or env sequences using an in vitro transcription kit according to the manual. It was synthesized by inhydro transcription of a DNA template. In short For example, 300 ng of the recombinant molecule corresponding to 924 was added to lysis buffer for RN4 acquisition (l guar). Nidine isothiocyanate, 125d sodium citrate pH 7,0,0,12 Solubilized in 5% sarcosine I., 50% dimethyl sulfoxide) and in the same manner. HIV inactivated with solubilized psoralen (2600ng, 260ng, 2 6 ng, 2.6 ng of 1124 vs. rjS) with a nitrocellulose filter. plotted above. For reaction with gag-specific or env-specific probes A separate filter was used. Prior to adding each probe, The nitrocellulose filter is hybridization/buffer i & (3X 5 SC150% formamide, 5X Denhardt solution, 150ug non-specific The heterologous RNA was incubated at 42°C for 2 hours. The filter is the probe Incubate overnight at 42°C. IX 5SC10, 1% SDS solution Wash thoroughly! The samples were dried, air-dried, and analyzed by autoradiography.

The autoradiogram for dot plot hybridization is shown in Figure 5. Ru. The gag probe alone reacts with the nucleic acids of the recombinant particle sample (panel A). versus panel B). In contrast, both probes reacted with busoralen-inactivated viral copies. responded to control. This data is based on the RNA of recombinant restored V-1 particles or gag. This shows that the env RNA is not packaged. this The conclusion is that the ga used to form the v-gag2 Vacunnia recombinant virus g gene or 1 ([■Recently quantified as a packaging signal for viral RNA) (Figure 5, panel C1). arrow).

These facts indicate that recombinant 11V-1 particles do not have the ability to replicate. There is. This conclusion is consistent with the long-term incubation of CD4 with recombinant HIV particles. “Intracellular endogenous 1 (described in section 12 indicating the absence of IV anti-antigen) Supported by experiments conducted.

7. Example Non-reproductive recombinant production using a single recombinant vaccinia virus vector H+V-i grains whose formation two recombinant vaccinia as described in section 6 Instead of a system for producing recombinant HfV-1 particles using a viral vector, HI Construction of recombinant vaccinia virus containing env and gagitfz of V-1 A system using a single virus hector containing a single virus can be used. In the example below, I (recombinant vaccinia virus containing the env and gag genes of IV-1) Used to infect BSC-40 cells. Infected cells are old V-1 envelopes These antigens aggregate to sensitize recombinant H[V-1 particles. It is formed within the stained cells. Recombinant old V-1 particles purified from infected cell supernatants were It was immunogenic in humans.

7.1. General method 7.2. Creation of v-G2E5 Containing HIV-1env and gagXt genes recombinant vaccinia virus The entire env code sequence under the control of the promoter in vaccinia virus 7.5 A 3.2 kb fragment (nucleotide numbers 5707-8608) containing the plasmid de pv-env 5 (U.S. Patent Application No. 071593.401. October 1990 Extracted from EcoR [filed on the 5th]. This fragment is the plasmid pv-gag It was inserted into the 2N EcoR1 site. (pv-gag 2N is a translation termination signal A synthetic linker with a U.S. Patent Application No. 071593401 (October 5, 1990), except that It is the same as pv-gag 2 described in Japanese Patent Application). ) Plasmid p~・-g ag2N is also under the control of the vaccinia virus 7.5 promoter) ) Contains the gag gene of V-1. Created plasmid pv-G2E5 The flI structure of is illustrated in FIG. The gag and env genes of HIV-1 are , described in U.S. Patent Application No. 071593.401 (filed October 5, 1990). As previously described, the thymidine kinase gene of vaccinia virus was isolated by in vivo recombination. Recombinant vaccinia containing the gag and env genes of old V-1 by introducing into the gene Create virus -=-G2E5.

7.3. Old ＼’-1 in BSC-40′a cells infected with v-G2E5 Co-expression of envelope and core antigens African Mitris monkey cells (BSC-40') were transformed into recombinant v-G2E5. Therefore, infection was carried out at a multiplicity of infection of 5 pfu/cell. By 24 hours of infection, cells were jc, and the cell lysates were separated on a 7-15% 5DS-PAGE gel. Thin Proteins from the cell lysate were transferred onto a nitrocellulose filter and human serum followed by goat anti-human immunoglobulin conjugated to horseradish peroxidase. treated with brine antibody. Immunoreactive proteins react with peroxidase substrates. Detected by reaction.

According to the results shown in Figure 7, both the envelope antigen and core antigen of old ■-1, v - Expressed and processed in cells infected with G2E5. of both antigens The amount of infecting a single recombinant vaccinia virus containing env or containing gag The amount was equivalent to that in the cells.

7.4. Recombinant H produced in BSC-40 cells infected with v-G2E5 rV-1F standing child Recombinant v-G2E5 was used to generate HIV-like particles from BSC-40 cells. It was. BSC-40 cells were grown using Cytodex 3 beads (Pharmacia LKB Bait). Otechnology) and supplemented with 5% fetal bovine serum according to the manufacturer's instructions. cultured in spinner culture bottles in Dulbecco's modified Eagle's medium containing . When the cells reach confluency, the recombinant vaccine is applied to the cells at a multiplicity of infection of 5 pfu/cell. The cells were infected with senior virus v-G2E5. After 1 hour of adsorption, cells were over-seeded. The cells were washed twice with fresh medium to remove any virus that may have been removed. 24 hours at 37,5°C After incubation, the medium was collected and cell debris was removed by low speed centrifugation. spare The clarified medium was heated at 19.00 Orpm for 3 hours in a 192 rotor (Sorva, II). and centrifuged. The precipitate obtained by this high-speed centrifugation was resuspended in PBS. , 32.00 Orpm for 1 hour using a 5W55Ti rotor (Beckpan) It was collected by centrifugation and reprecipitated. Resuspend the final level in cold PBS. and stored at -70.5°C until use. 924 and gp12 of the prepared particles 0 antigen essentially described in Section 6.1.2. p24 respectively as described in Antigen capture and immunoprocedural assays were used.

Radioimmunoprecipitation analysis of particles produced by cells infected with v-G2E5 is shown in FIG. These results prove that both high-speed centrifugation and This suggests that these mature pirion proteins selectively assemble to form particles. It shows what will happen. The relative amounts of p24 and gl)120 in these particles are H Similar to the relative amounts in rV-1 pyrion preparations, the difference between recombinant and authentic HIV-1 Showing structural and biochemical similarities between pyrions.

8. Example: Non-replicating recombinant H produced by introducing plasmid DNA into mammalian cells Formation of rV-1 particles Using the recombinant vaccinia virus vectors described in sections 6 and 7, In addition to the system for producing recombinant H[V-1 particles, HtV-l env and gag genes A system containing mammalian cells into which DNA encoding the gene has been introduced can be used. this The system can be used to produce recombinant H[V-1 particles in stable mammalian cell lines.

Recombinantly produced H[V-1 particles using the system described here are listed below (this (including, but not limited to) various methods.

fl, l Composite plasmid vector containing many HIV structural genes or control genes transfection of tar (2) Simultaneous replication using multiple plasmid vectors containing different HIV genes infection +3) Constitutive and regulatable enhancers that promote HIV protein expression /Using promoters (To indirectly control the expression of gag and env structural proteins of 41H[V Use of HIV control proteins containing tat and/or rev (51 HIV protein) Use of heterologous cell lines for expression of proteins and recombinant HIV particles (6) Use of a plasmid vector encoding a modified old ■ protein.

These variations allow optimization and assembly of the system in different cell types. Manipulation of different protein species or structures within engineered HIV particles is possible.

8.1. Plasmid construct Many of the plasmid vectors used in this system include the expression vector pH31JPy (, Aruff and 5eed, 1987. Proc, Natl, Acad, Sci, U, S.

A, 84:8573-77)-derived hybrid CMV: old ■ enhancer - Contains a promoter (named CmHi), pH31JP y is 1 ([V promoter and tar region (nucleotides -69 to +78) Contains an enhancer derived from the combined cytomegalovirus immediate gene . Some of these plasmid vectors are cytomegalovirus immediate gene (named CMV) or another derived from the mouse metallothionein-■ gene (Mt). Contains enhancer-promoter elements. These control elements It is linked to the gene sequence encoding the protein, and the polyA of the Ela gene is attached downstream of it. Hpal-Hhal fragment of adenovirus 2 containing the additional region (nucleotide 156 9 to +680), and further downstream of that, BamH[- The 3ph1 fragment was connected and the plasmid Bluescribe plus cloned into Tegene). coated by each of these plasmids The respective HIV proteins are shown in Table I and these plasmids are shown in Figure 9. Illustrated.

(Margins below this page) HIV gene vector encoded in a plasmid vector (below T-margin) Blasmi)” CmHiEnv5 (1104-bl) is a cytomegalovirus. Immediate gene enhancer, HIV promoter and +ar? including X Circle 13NIP5- connected to 567t-8572Avall-Kpn[ It has a Nrul-Hindlll I fragment.

Plasmid CmHiTgfbEnv5 (1113-al) is a chimeric TGF-β Nrul-Pst[env] of pH31JPy linked to H[V env gene. Contains a promoter fragment. In addition, 5゛ untranslated region and signal Signal cleavage of the monkey TGF-β gene or old V env gene that supplies the peptide Kpn from nucleotide 5856 to nucleotide 8572. It is located at site I.

Plasmid CmHiGag2Rre (1158-al) is a former Vgag and pol )-to the Bs5H[r site at nucleotide 257. Nrul-Xba of pH3MPy located at the Asp718 site of Enhancer m: Contains a promoter fragment. This is a recombinant vaccinia virus This is the same region as the gag coding sequence contained in v-gag2, and the entire gag leader contains the reading frame and about half of the following <pol reading frame. Ru. In addition, this region contains a sequence that coats the old protease and a reverse transcriptase. Contains most of the arrays to be coded. Xba I linker that provides translation termination futon teeth! 5p718 site. Ba at nucleotide 7178 downstream of it Hindl[I contains the Rev response element at nucleotide 1''7698 from 1lI. The HIV env gene fragment is established. Plasmid CmvGag2Rre (1 159-al) is NruI derived from the ruler vector CDM8 (Invitrogen) - Using Xba l cytomegalovirus enhancer and promoter fragments They are the same except that they are

Plasmid CmHiRev (1132-cl) contains Bs of nucleotide l'5500. Kpn1 site (nucleotide F') from u361 site to nucleotide 8572 site 5590-7935 is in the process of being removed), and H[V pi (3MPy Nru I-Ps Junha) located upstream of the Rev gene Contains the 7 sir promoter fragment. Plasnide Cmt・Rev (t15 2-at) is Nru[-X Do not use haI cytomegalovirus enhancer and promoter fragments. They are the same except for

Plasmid CmHi Tat (1132-bl) contains S at nucleotide 5331. Kpn at nucleotide 8572 from al[site (nucleotide 5590 -7935 has been removed) and has no intron Nru[-Bgll! of pH3111Py upstream of the gene. Enhancer Bufumo Contains data fragments.

Plasmid BsMtRev (1202-2) contains one transcription initiation site. Mouse metallothionein-I gene starting from EcoR1 site located at 700 Contains a 1.7 kilobase fragment of HIV that does not have sotl and it introns Located upstream of the ev gene, there is a nuclease from the Bsu36 site at nucleotide 5500. Kpn [site of leotide 8752 (nucleotides 5590-7953 removed) located in ).

Plasmid Bs! JITat (1203-1) is located approximately -170 upstream of the transcription start site. Mouse metallo oneine-I starting from the EeoR1 site located at 0 (Tachiko's Contains a 1.7 kilobase fragment. It is H[V t:a, which does not have an intron. Located upstream of the gene, from the Sal1 site of nucleotide 5331 to the nucleotide Kpn of 8572 [located at site.

Blasmi FCmHIVdelXmn (1133-at) is a previously described protocol. Expression vector pH3 used in lasmid! JPy Yoshison's hybrid CL IV: Contains the HIV enhancer-promoter. This plus: 1・′ X at nucleotide 384 within the N-terminus of the beetle gag reading frame mn [H in the part [V5’ leader I? An NA sequence has been inserted. within this fragment Many of the former genes encoding the tat, r'ev and env proteins The splice spliced to the acceptor site located upstream of the H[V gene Contains one part of Price Tona. Xmn at nucleotide 384 [site is Xb ai linker (translation of gag N-terminal peptide at approximately 20 amino acid residues) pal reading frame (including the TAG translation stop codon that terminates the The XmnI site at nucleotide 4034 near the C-terminus of the protein. H The rV sequence is the vif, vpr, tat, rev, and vpu proteins of HIV. The former ■ control region and minor structural proteins including H[V env protein Following the Kpni site at nucleotide 8572, I'm there. This plasmid consists of a 5-splitase located just before the N-terminus of the gag gene. Upstream of the rice donor site and reading frames encoding various proteins By splicing that occurs between the splice acceptor site located at List above; each I ([V protein should be encoded. .

Plasmid CmHiVdelKpnAvr (Gag2TRE) (1160- al') contains sequences encoding tat, rev, and env proteins The entire gag reading frame and pa directly linked to the 3' region of the old V genome. The reading frame of the N-terminal region of l (recombinant cotton cilia virus v-ga Hybrid CM■~old■enhancer that causes the expression of (contained in g2) - Contains promoters. In this plasmid, the expression vector p3L IPy cytomegalovirus immediate W”, IJl (E child enhancer-fragment N ru[-EcoR[ is located in the HIV promoter sequence starting at nucleotide -69. are combined. The HIV leader RNA sequence passes through the gag gene to nucleotide It continues to the pot gene up to the Kpn 1 site of 3372, and the pot leader The polyline containing the Nhe1 site, which includes the TAG translation stop codon in the translation frame. connected to the linker. This Nhe [site is within the vpr reading frame Avr I at nucleotide 5207 [binds to the site. H[V sequence is null Kpn 1 site of leotide 8572 continues. This vector is HiV ag, protease, tat, reV, and en~ protein synthesis, considered important for pricing, intracellular trafficking, translation, processing and function Contains all array elements that are This plasmid is a gag gene (N- A 5' splice donor site located just before the end and a site encoding various proteins. splice acceptor site located upstream of the reading frame Due to splicing that occurs, unspliced mRNA is Each H [■ should be able to encode the old Vgag for the protein. be. It is the first 60 nucleotides or more of the first 'EA' mRNA transcript of H[V. ta, which is located within the HIV promoter and is required for tat trans action from the HIV promoter. r element and located between nucleotides 7315 and 7559 and is catalyzed by rev H[■Rre required for intracellular localization of mRNA encoding structural proteins ( rev reaction element). This plasmid vector contains reverse transcriptase The C-terminal half of the pal reading frame, including part and all of integrase. minutes, N of all V1f and vpr reading frames - The central region of the H[V genome encoding the end is not included. Also, this vector - includes most of the nef reading frame, the main part of the 5' LTR, and the 3' L Not all of the TR is included.

8.2. Recombinant HIV-1 pellets from stable CHO cell transfectants Formation of offspring Plasmid vector CmHIVdelKpn, Avr (Gag2TRE and CmH iEnv5 was transfected into dhfr-CHO cells. transfection cell lines were co-transfected with plasmid pSV2dhfr. Selected. CHO) Transfectant produces immunoreactive mature gag and env Contains proteins such as HIV pillion and those listed in Section 6. Recombinant restoration V formed according to the above! in the form of particles with sedimentation properties similar to particles. secreted.

8.2.1. Transfection and selection of cells 10 in 60 mm tissue culture dishes Ham's F supplemented with % fetal bovine serum and 15 μg/ml proline Grow to 50% confluence with 12 nutrients (no hyboxanthin), serum free Transfer to culture medium, ribofectin (BRL), H[V expression plasmid CmHI VdelKpnAvr (Gag 2TRE) (1160-al) and CmHi Env5 (1104-bl) mixture and selectable plasmid pSV2d 5 hour incubation with hfr, lipofectin:DNA mixture CHO cells (dh fr-) was transfected. 2-3 days after transfection, culture The culture was transferred to selective medium DMEM supplemented with 10% FBS and proline. 2 days later Trypsinize the cultures and add aliquots containing approximately 0.08% and 0.40% cells. Recoat (containing approximately 1 and 5X!0' transfected cells, respectively) (estimated to be r) was transferred to a tissue culture well with 96 wells. 7 ．． Aliquots containing 8% cells were transferred to 6-well tissue culture dishes.

After culturing for an additional 13 days in selective IR medium, one set of 6-well Brake 1 was fixed. and incubated with AIDS patient serum (TrilJar), then Rumor peroxidase conjugated goat anti-human antibody and peroxidase substrate (3- by incubating with amino-9-ethyl-carbazole). Cells were immunostained for protein expression. 15-20 per well Colonies of selected cells were present. According to immunostaining assays, approximately 10% of These colonies were found to have HrV protein. These analyzes is only about 10 to 15 cells per plate seeded with fewer cells. wells contain colonies of transfected cells, and both of these wells contain colonies of transfected cells. showed that the HrV protein is likely to be expressed by the HrV protein.

Tissue culture media from individual wells of 96 L plates were collected and ga Secreted gag protein was assayed with g protein antigen EIA (6. ．． Section 1.2, supra). A large number of II wells were identified by this assay. 12 pieces potentially positive wells, from each of three independent transfections. Four wells were selected for expression. Visually examine positive wells. Then, some wells are confluent in nature and those in the pond have small proliferating cells. They only have colonies, and those in ponds clearly contain visible cells. did not exist. Cells of all 3gI classes in potentially positive wells were grown. . : Trypsinize the wells and place the cells in a 6-well dish for expansion. was moved.

A small fraction of cells from each candidate cell line or in duplicate wells of a 24-well plate. was inoculated during the course. After 5 days of growth, wild-type HeLa cells or HeLaT 4 cells transfected and expressing CD4 protein) were added to each well. added. The next day, gag expression and envtl capacity were upregulated by focal immunoassay. Said. Briefly, cells were fixed and then incubated with human anti-HIV serum. horseradish peroxidase-conjugated goat anti-human antibody and peroxidase enzyme substrate (3-amino-9-ethyl-carbazole). 2nd group Four separate cell lines were positive in this assay. Two cell lineages ( 3010-CI and 3010-C6) have only a few cells or are very strongly stained, and are different from HeLa T4 cells in large syncytia (sy ncijia) or not with wild-type HeLa cells. . Two pond series (3010-C5 and 3010-C13) had large numbers of positive cells. These were more weakly stained than those mentioned above, with only one fraction HeLa T4 cells and syncytia were formed. Cells from other lineages are stained It wasn't done. Antibody staining of these positive cells indicates that they synthesize gag protein. This suggests that HeLa and T4 cells form syncytia. These are the functional gp+20 and gp old envelope proteins on the cell surface. This indicates that the protein contains a proteinaceous substance.

After further expansion of each of the candidate cell lines, ll+V protein synthesis was determined. Cell lysates were analyzed by Western blot. 60mm or 100mm Cells in tissue culture blood were washed twice with PBS and directly placed at 300 μm or 750 μff. Drain in Laemmli sample buffer. After boiling, all cells in the sample Proteins were run on 1006 polyacrylamide gels or on 8-16% gradient polyacrylamide gels. It was resolved by electrophoresis on Rylamide gel. CEM@n that infected old ■ and old ■ I cells, vaccinia recombinant v-gagl, v-gag2 or v-env5 An aliquot of infected B5C-40 cells was included as a control. of gel The contents were electrotransferred to a sheet of nitrocellulose filter. The filter is , rabbits immunized with gp160 from AIDS patient serum or v-env5 infected cells. After reacting with one of the serums of the 125-I and The mixture was reacted with Fin A and washed thoroughly again. Autoradiate the product with X-ray film Visualized using ophography.

The four positive series identified in the above assay were again associated with the gag protein. was found to be positive. 3010-CI and 3010-C6 Zeite has a number (g more than 3010-C5 and 3010-("+3 lyzate) It contained gag. Lines that are negative in the above assay are also Stamplot was negative for gag. Many of the immunoreactive substances are p 55 precursor and in or by HIV or v-gagl. It is co-translocated as a slightly smaller species that does not represent the main product in infected cells. Ta. Extremely small amounts of mature p24 and p17 gag proteins are present in cell lysates. It was detectable in the test. This analysis also shows that g l) 160. gp120 and gp41 envelope proteins are present Which indicates that.

8, 2.2. Characterization of recombinant HrV-1 particles synthesized in these cells. gag and env proteins are assembled into virus particles and secreted. To demonstrate, tissue culture supernatants were collected and clarified by low speed centrifugation. Both parts of the particle are far away Collected by heart (32,000 rpm with SW550-tor, or SW old rotor (either 27,000 rpm with a 19-inch rotor or 19,000 rpm with a 19-type rotor).

Western blot analysis showed that these substances inhibited gag and env proteins. It was shown that it contains both. The main gag protein detected in the particles is Mature p24 and 917 species: smaller amounts of p55 and p40 precursors can also be detected. Ta. In contrast, precursor species accounted for much of the material in cell lysates. gi+ 160. gp120 and gp old envelope proteins are all detected in the particles It was done.

Comparison of Western blot h shows small fractions of gag and env proteins contained in cells. This shows that only proteinaceous substances are secreted as particles. derived from a large number of 30!0-C6 cells. Quantitative gag antigen EIA analysis of particle preparations from 3 to 17 ng per ml of medium. gag yield. Particles from the 3010-C6 cell line were also tested on sucrose gradients. (2 hours at 50.00 rpm in a SW550-tar, 15-60% sucrose). As shown in Figure 10, fractions from the gradient were When both raw EIA and Western blot assays are performed, the particles are approximately 35-40 % sucrose showed that a single peak band was formed.

8.3. Ike's strategy for env and gag protein expression and mammalian Recombinant HIV-1 and particle formation in cells In this section, we will discuss the formation of recombinant HIV-1 and particles in transfected cells. Other studies regarding the expression of HIV-proteins and recombinant HIV particles in It! A number of variations for representing abbreviations are described. Possible or not limited Variants include: (1) Numerous HIV structural and/or regulatory genes Transfection of complex plasmid vectors containing (2) different HIV Co-transfection of multiple plasmid vectors containing genes, (3) Phacogenic and regulatable enhancers to drive expression of HIV proteins /Use of both promoters, (4) HIV gag and env structural proteins Contains tat and/or rev to indirectly control quality expression (5) the use of regulated expression of Hlt” regulatory proteins in different cell types. Expression of H1■ protein in. These variants are based on various parameters of the system. Useful in optimizing protein and particle size in different cell types Allows independent manipulation of components and expression levels.

Blamid and plasmid were isolated by standard calcium phosphate transfection method. HeLam cells (and in 2-3 cases, tested by transient transfection into B5C-40 and Veram cells). Ta. The product is a whole cell lysate or a recombinant product collected from the culture medium by high-speed centrifugation). IIV - Particulate polyacrylamide gel electrodynamic, nitrocellulose filter and analysis by electrotransfer to and probing with specific antisera. env To analyze proteins, filters with gl+160 and gp+20 A +25-T labeled mono that binds to an epitope in the vSfri region. + gag tan searched with clonal antibody +10-4 (sections 6, 1, 2, above) To analyze protein, the filter was filtered with AIDS patient serum (TriMar), Next, a search was performed using protein A labeled with +25-1. The product is Visualization was performed by film autoradiography.

111.3.1. Expression of HrV protein in HeLa cells is expressed by a number of different Obtained from plasmids and combinations of plasmids.

Expression of the former 'V gag and env proteins is both a structural and regulatory protein. Transfer of a complex plasmid encoding multiple HIV proteins containing Alternatively, different) l[V protein Multiple plasmids encoding the quality may be co-transfected.

plus that codes env (CmHiTgfbEnv5 or CmHiEnv5) A plasmid encoding a plasmid encoding mid or gagCmHiGag2Rre) is In combination with plasmids encoding r and rev proteins, HeLa cells When transfected with immunoreactive gp160 and pg120 ■Produce expression of envelope proteins or p55-translation products, p4 7 and p39 brosesong intermediates, and P24 and p17 mature gag proteins. resulting in the expression of immunoreactive HIV gag-related proteins, including.

On the other hand, within a single transcription unit, tat and rev regulatory proteins and enV, or , functional coding sequences for both env and gag structural proteins. (Cm old VdelXmn or CmHIVdelK pnAvr (Gag 2TRE) was transfected into HeLa cells. synthesis or production of both precursor and mature env or gag and env proteins. Jiru. CmHIVdelXmn as CmHiGag2Rreg or CmVGag2 Co-transfection with Rre also induces gag and eny proteins. Bringing about the synthesis of both qualities. Each plasmid or their combination CmH[Vd elkpnAvr (Gag 2TRE) (+160-al), CmHIVd elxmn (1133-at) + CmHiGag 2Rre (1158- ari, and CmHIVdelXmn (1133-at) + CmvGag 2Rre (1159-al) also induces secretion of I-modified H[V particles into the medium. bring about.

Thus, gag and env fl structural proteins using this system Expression and production of recombinant HIV particles is accomplished through multiple steps. different A comparison of the levels of expression achieved by combinations of plasmids is shown in Table III. This makes it suitable for different combinations or expression of different proteins. It shows. For example, env encodes the tat and rev proteins separately. Expression is more efficient when co-transfected with a plasmid that It seems like that. On the other hand, gag is a mechanism for both regulatory proteins within a single transcription site. CmHtVdelKpnAvr (Gag2TRE), which contains a functional coding sequence. (1160-al). both Intermediate levels of protein in the complex plasmid CmHIdelKpnAvr (Gag2TRE) (1360-al) and separate erlV: l-do conversion plus Obtained by co-transfection of MID.

(Less than 100 margins) Table ■ Using different combinations of plasmids (env and gag protein Relative level of quality expression plasmid env gag CmHiEnv5 + Cm) IiTat + CmHiRev +++++Cm HiGag2Rre + CmHiTat + CmHiRev -+10Cmv Gag2 + Cmv [? ev +Cm)llVdelXmn + CmHiGag2Rre + CmH[VdelXmn + 10+++Cmt( IVdelKpnAvr (Gag2TRE) +++++CmHiEnv5 + CmHIVdelKpnAvr (Gag2TRE) +++ +++ gag and plasmids obtained after transfection with various combinations of lasmid Relative expression levels of gag and env are shown at arbitrary sites; gag and env expression Levels are measured independently and are not necessarily expressed on the same scale.

In these experiments, rev is I ([mR encoding the Vfll-forming protein Important for efficient cytoplasmic localization of NA, the N-terminal region of the gp41 molecule It is localized within the HIV env gene portion that encodes the rev response element (Rre ) and is called ga as a BglII-Hind■ fragment from the envelope gene. g plasmid and relies on cis-acting regulatory elements introduced into the HI Is it essentially necessary for efficient expression of V gag and env proteins? Maybe. The tat protein contains the first 75 nuclei of the H[V RNA transcript. present in cleotides and interacts with a stimulant-acting regulatory element called tar. [(Important for increasing transcription initiated from the IV promoter) It is. The tar gene is CmHiGag2Rre, Cmt(iTgfbE Cm old hybrid enhancer used by nv5 and CmHiRev - required There is r7 inside the element.

Expression of gag or env is similar to C+nvGag2Rre and CmvRev. Can it be done independently of tat by co-transfection of plasmids? I'll come. This is because these plasmids do not contain a tarfa region. Ru. However, the transfer of CmvGag2Rre + CmvRev gag expression from CmHiGag2Rre+Cm)IiTat- lower than that from CmHiRev transfection. site megalow The enhancer promoter of the viral immediate gene is an extremely powerful transcriptional element. Therefore, this means that 1 (IV tat l!den and CMV: H [V enha Combining sensor and promoter elements creates a particularly powerful expression system. Which indicates that. Similar observations were made in the context of env protein expression.

Other enhancers required! ,! = Hybrid between HIV promoter/lar element The net may have useful properties in the pond.

These results differed in that (1) structural proteins were differ in their need for HrV regulatory proteins or other elements, and e by selecting a corresponding plasmid or combination of plasmids. This shows that the ratio of gag to nV protein can be controlled.

These results also indicate that plasmids encoding different old proteins or independently This shows that it can be introduced into cells. As one example, recombinant H[V particles The cell line producing the recombinant particles may change the manufacturing level and/or the properties of the recombinantly produced particles. Because of this, minor H[V proteins such as vpu or vif The lasmid can be further transfected. As an example of a pond, fat, re Cells transfected with plasmids encoding V and gag proteins series, which therefore produces particles containing only gag protein. selected to contain env proteins from different strains or different env-encoded plastics to produce particles with structural changes in the pond. Lasmid transfection is also possible. For example, HIV en ~ gene (C The natural signal sequence of Env5) or the signal sequence of monkey TGF-β-1 One of the fusions between the column and the N-terminus of mature gp160 (CmHiTgfbEnv5) Comparing the expression of env protein from genes containing either A rapid onset was observed.

8.3.2. HIV er+t protein through the use of regulatable promoters Regulation of expression In other embodiments, the dhfr-CHO cells contain the selectable plasmid pSV2dh CmHiTgfbEnv5 + CmH, which combines plasmid with fr iTat+CmHiRev transfected. selected in this experiment Initial testing of selected cell lines was performed to determine possible levels of HiVer+v expression. I couldn't. Cells were then transfected with selectable pSV2 Amplification of the dhfr plasmid and encoding the old V rat, rev and env in increasing levels of methotrexate for co-amplification of plasmids. This is a low-level increase in HIV env expression. There was something you would expect to choose width. l [V env protein is synthesized One cell lineage previously identified was confirmed. However, the dhfr gene and simultaneous For amplification of a line of transfected old protein-encoding plasmids. Selection by proliferation at gradually increased methotrexate C did not result in expression of env protein. Rather, the envelope tamper protein expression levels decrease in cells grown without niscarating selection. Therefore, continuous selection is necessary to maintain env protein expression levels. there were. Cells expressing high levels of env protein also have lower levels more slowly and better than cell lines that reverted to full expression of the env protein. appeared to grow to lower densities.

These results suggest that envelope protein expression or toxicity may be the cause in these cells. , therefore suggesting that there is a limit to the achievable level of enV protein expression. are doing.

This limit is due to the regulatable promoter that controls expression of the H[V protein. This can be affected by the use of plasmids that utilize

Such plasmids are first transfected into cells grown in non-induced conditions. , which can then induce high levels of HIV protein expression; It allows for high levels of production in a short period of time before more toxic effects occur.

As an example of such an approach, metal-modulated mouse metallothionein-■ Plus coats the tat and reV proteins, respectively, under the control of the promoter. Midoka was constructed. Combination of HeLa cells and plasmids: (a) CmH iTgfbEnv5 + CmHiTat + CmHiRev.

(b’) CmHiTgfbEnv5 + Bs! JtTat + CmHiR ev and (C) CmHiTgfbEnv5 + CmHiTat 10BsM tRev, co-transfected with i>, then whole cell lysate was Tan plot analysis showed that immunoreactive gp160 and gp120H [It was shown that V enherobe protein was produced (Fig. 11). axis) 1i Transfection with TgrhEnv5 + CmHiTat + CmHiRev tion produced the highest level of env protein expression. Even more meaningfully For transfection using the Mt promoter, the tissue was harvested one day before harvest. It was found that adding zinc to the culture medium induced the expression level several-fold. (See Figure 11).

These results demonstrate that regulatable promoters regulate the expression of HIV proteins. and the expression of HIVI synthetic proteins can be used for) 11vtar and re It has been shown that it can be regulated indirectly by modulation of v regulatory proteins.

A good example of this approach is to create complex plasmids encoding multiple H[V proteins. Use of regulatable promoters to control expression from For example, a metallothione protein linked to the promoter and tartif region of H[V Hybrid Mt containing Romotor metal adjustment elements: Hi/Enhance including the construction of novel inducible regulatory elements such as promoters.

8.3.3. Expression of H[V protein by B5C-40 and Vero cells For example, CmHiGag2Rre + CmHiEnv 5 + CmH HiV fat, rev, en containing both iTat + CmHiRev Combinations of various plasmids encoding the v and gag proteins, and Cm H[~’delKpnAvr (Gag2TRE) to HeLa, B5C-40 and transfected into Vero cells. Basic pattern of expression of gag and env The vector is divided into three cell lineages, both in terms of what is produced and the efficiency of expression. However, the overall expression is similar to the tteLa details. B5C-40 cells have the highest number of cells, 3 times lower in B5C-40 cells, and even lower in Vero cells. 51S low.

These results are consistent with the previous results for C1(0 cells) in different species and Different cell lineages derived from different organs ([used to express the V protein) cells with specific desired properties or for special applications. This shows that it can be selected.

9. Example, recombinant vaccine expressing truncated H[V-1 envelope j Recombinant HIV- Formation of 1 particle of truncated HIV-1gp1 in infected B5C-40 cells. Two recombinant vaccinia viruses directing the expression of V. 60 are described below.

These recombinant vaccinia viruses use the system described above in Section 6. to provide recombinant HtV-1 particles with enhanced anti-viral and/or immunogenic properties. Producing Tanuni, v-gag2 (Section 6 and the next section, above) or other core antigens Vine used as a vector along with code vector.

91, B downstream from the promoter in recombinant vaccinia virus V-ED27.5 Nucleotides inserted into Wachnonia recombinant vector pGS62 at am81 site Old V-1 env that coats sequences numbered 5705 to 8068 (continued) US Patent @1￥07/’593°401, filed October 5, 1990 ) was constructed horizontally. HIV river sequence sequence lasmid pv-en It was derived from v5 as a 2.36 Kbp BamH1 fragment (19 Pending U.S. Patent No. 07, filed October 50, 1990. = '593.401). Cut off The fragment contains the entire coding sequence of gp120 and the N-terminal 241 amino acids of gp old. 49 of the cytoplasmic region of gp old at the C-terminus of the proposed transmembrane sequence. It had the amino acid residue g1. Application filed on October 5, 1990 and ongoing 7.5 according to the method described in U.S. Patent Application No. 071,593.401. A chimeric gene containing the promoter and old V-1 env sequences was isolated from vaccinia virus. inserted into the gin kinase gene.

The resulting recombinant virus v-ED2 was as efficient as wild W gplFio. It directs the expression of a truncated form of gl”+60 that is cleaved into gp120 and gpold. (Fig. 12), lela CL” cells (infected with v-ED2) are full-length easier than v-env5 expressing gp+60 envelope glycoprotein precursor Formation of cystium (giant cells formed in polycrystals, characteristic of HIV-1 infection 61) Ru. These results demonstrate that the envelope glycoprotein produced by v-ED2 than the “wild type” envelope glycoprotein! functionally more active, Or, it shows that it can exist more effectively.

9.29 Recombinant vaccinia virus v-ENV5DCTgp old C-terminal 13 H[V-)v (BRU-isolate: Wain-Hobs on et al., 1985. All env code layout of Ce1l 40:9317) Recombinant virus v-ENV5DCT was constructed to include columns. (Kunk el et al., 1987. ue+h, In Enzymol, 154: 367-382) by oligonucleotide mutagenesis methods. A deletion mutation was introduced. Filed on October 5, 1990 and pending in the United States Promoted to 7.5 according to the method described in patent application no. 071593.401 Soniawi, a chimeric gene containing a mutated HIV-1 env sequence inserted into the lustimine kinase gene.

10. Example: Recombinant vaccinia expressing unprocessed gp160 A virus was used to generate recombinant H[V-1r1 progeny possessing decorated structural characteristics. g with a mutation in one proteolytic cleavage site between gp120 and gp41 Two Recombinant Vaccines Directing Expression of the pI60 Precursor Envelope Glycoprotein Near virus is described here. Using the system described in Section 6 above To produce recombinant H[V-1 particles that can enhance antiviral and/or immunogenic properties. These can be used together with v-gag2 or other Fur antigen-encoding vectors to recombinant vaccinia virus can be used as a vector.

+0.1. Recombinant vaccinia virus v-16ONC vaccinia recombinant vector - Nucleotide number inserted downstream from the promoter at 7.5 in pGS62 Recombinant plaque containing mutated gp+60-coat sequences from 5803 to 8495. Sumido (pl・-16ONC) was built horizontally. (Kunke I et al., +98 7°! Jeth, Enzymol, 154: 367-382. Mutations are introduced by oligonucleotide-directed mutagenesis (as described). It was done. The mutated sequence is indicated as follows (“ is between gp+20 and gp41 (represents the cutting site) (hereinafter referred to as the margin) HIV-18Renv gp120 gpJIGfn Arg Glu Lys Argf Ala “wild type”, CAG AGA GAA AAA, A GA f G irises...

v-+6ONc,,, CAG mGAA UAA m ↑ GC 8,,.

Gln Ite Glu Glu Phe ↑ AlaOn October 5, 1990 As described in pending U.S. Patent Application No. 071,593°401 By in vivo recombination, chimeric genes or mutants were generated in the thymidine kinase gene. inserted into the Cuccinia virus genome. The obtained recombinant virus was gpl! 0 directs the expression of the +60 precursor envelope glycoprotein (gl) which is not cleaved to command.

10゜21 recombinant vaccinia virus v-11! , 6ONc (section 10,1, previous ) Does it contain the same mutated) IIV-1env gene as v-16ONc? , a recombinant vaccinia virus under the control of the vaccinia virus IIK promoter. Rus or built. 2.26Khp BamHt fragment or plasmid pv-160 Vaccinia was excised from the NC at the EcoR1 site downstream from the IIK promoter. derivatives of the recombinant vector pscIO (Chakrapati et al., 1985. o1. Ce11. Biol, 5:3403-3409) was inserted. child This contains the complete code for the vaccinia virus IIK promoter and mutated gp160. Generates chimeric progeny containing the sequence. This chimeric gene is called thymidine It was inserted into the vaccinia virus genome at the enzyme gene. The generated recombinant virus rus directs the production of high levels of mutated H[V-1gp+60 in infected cells. command.

11. Example: Recombinant vaccinia virus expressing old V-1vpu gene The construction and production of recombinant body V-1 involves the use of HIV-1 envelope and core antigens. No extra HrV-1-specific proteins are required. However, due to the virus Other elements of cell and cell origin can be involved and facilitate this method. Ru. One such element is the viral protein encoded by vpu It is. This protein is a +6Kd glycerin that is apparently associated with the inner surface of the cell membrane. It is a non-codylated polypeptide. vpu is not required for particle formation Alternatively, mutations in vpu result in decreased pyrions released from infected cells. (Terwilliger et al., +989, Proc, Natl, Aca d, Sci, IJ, S, A, 86:5163-5167; 5trebel et al. , 1989. J, Virol.

63:3487-3791). The mechanism of vpu action is not clear and is described here. The role of particle formation in such recombinant systems is not clear.

Auxiliary molecules such as vpu may play a role in facilitating the process of pyrion assembly and/or release. It is hoped that it will be able to fulfill this role. The following examples demonstrate the use of such auxiliary molecules. The potential role of these systems is discussed and their use in recombinant particle formation is demonstrated. Describe the program.

Does the recombinant vaccinia virus contain the extra V-1vpu-coding sequence? +a was built. H [V-1 cDNA 2 A 90-bp fragment between Sma[ and EcoR[ 7.5 downstream from the promoter was inserted into the Vaxonia recombinant vector pGW62. This fragment is 7 bp of 5'-untranslated sequence and 37 bp of 3-untranslated sequence. ing. Next, chimeric genes are added to the vaccinia virus genome by in vivo recombination. A child was inserted. The effect of vpu on the formation and release of recombinant particles was discussed in the previous section. As described in Section 7 and subsequent sections, v-vpu and V-G2E5 can be demonstrated in co-infected cells.

12. Example 3 Antiviral effect of recombinant old V-1 particles This example demonstrates the antiviral effect of recombinant old V-1 particles. Recombinant Old V particles reduce the infectivity of CD4” lymphocytes by HIV in vitro. , or the ability to eliminate. The results show that recombinant Old V-1 particles have a dose-dependent effect on the lips. It has been shown that this method effectively suppresses old V-1 infection.

! , 2.1. Infectivity assay T-lymphoblastoid cells (CEM) were grown at 4XIO' cells/unit in 0.4 ml of medium. 24-well culture plate (10% fetal bovine serum) RPM[-1640] supplemented with. Then, in duplicate wells, add 0.4 ml of medium. of recombinant HIV-1 particles (see Table 1) were added. The added particles are t determined by ERA and quantified by equivalent p24 gag concentration (see above, Section 1). 6.1.2. section). Control wells received no particles and 0.4 ml of medium. Only the volume was corrected. Cells and particles were incubated for 3 h before adding H[V. Incubated at 7'C. of duplicate wells corresponding to each of the various particle concentrations. One set is (1) H[V not added, (2) 5pg p24 gag 40TCID, corresponding to . Addition of low virus (50 tissue culture infectious dose units) Or (3) 400 TC equivalent to 50 pg p24! Ds. high virus The addition of Three days after infection, remove the medium from the wells and remove the corresponding original particles. Replaced with new medium containing concentration. On the 5th day of infection and on the next 6th day, all wheat Aliquots of cells were collected from the wells and treated with human polyclonal anti-H as secondary antiserum. ) using goat anti-human serum conjugated with fluorerasen as a secondary serum. Using an indirect immunofluorescence assay with both IgG and H[V antigens, Infectivity was assayed by measuring intravesicular expression.

122, Conclusion The infectivity assay results are listed in Table 1 below, and are Previous V-1 particles induced dose-dependent susceptibility of CELI cells by HIV pillions. This shows that it can prevent staining. With high concentrations of recombinant HIV-1 particles Since no intracellular fluorescence was detected in the cultured cells, the recombinant particles themselves It can be concluded that it is non-infectious and does not replicate in cells (Fig. 13, Panel A). Additionally, multiple syncytia were observed in any cell culture supplemented with HrV. Among other things, it was observed that the recombinant particles did not induce any noncytium formation. Ta.

Table ■ Inhibition of V virus infectivity + 60 0 0 by recombinant H[V-1 particles 0 +600 0 0 0 + 0 40 20 > 90 + 60 40 10-20 > 90 + 600 40 <1 10 + 0 400 90 > 90 + 60 400 60-80 > 90 + 600 400 30 > 90 1. The fold excess of particles against viruses is Relates to equivalent amounts of p2-1 protein.

2. Human immunodeficiency virus infection (HIV-1), L4V-1 strain 13, Example 8 Recombinant H[Vi particles are culture media isolated from former V-1 seropositive donors. Inhibits HIV infectivity of cultivated peripheral vascular lymphocytes.

The examples listed in Section 12 above show that recombinant, engineered shell V-1 particles were dose-dependent. Inhibiting old-1 infection of T-limboblastic cells (CEM) in vitro in a manner that It shows. The following example describes a cultured peripheral vascular lymphocyte (PBL) system. to confirm that the recombinant 52HrV-1 particles have antiviral activity. Ru. The PBLs used in this system were seropositive cells infected with HIV-1. The gradient of infected cells in both isolated PBL segments (fr (Psallidop) ranged from 0.04% to 1.3% (Psallidop oulos, M., C. et al.

1989, J. Virol, 631: 4626-4644). During cultivation In most cases, viral infection is cell-free, or cell-to-cell transmission leads to unstained CD. 4” cells. Recent findings from other groups (Daar, E., S. ,and others.

1990, Proc, Natl, Acad, Sci, U, S,, A, 87:6574-6578),) The laboratory isolate of IIV-1 was The effects of antiviral agents such as neutralization and soluble CD4 It has been suggested that there is a different response to this. Therefore, recombinant barrel V-1 particles Using a Cultured Peripheral Vascular Lymphocyte System to Evaluate the Antiviral Effect of It is biologically more efficient than the CEIJ cell system described in Section 12 above. This could be a more appropriate system.

! 3.1. Infectivity assay Iypaqueficoll cusion) Fractionation of the buffy coat material above allows peripheral vascular lymphocytes (PBLs) or HI Collected from heparinized blood samples of V-1 seroboditive donors. Thin The cells were incubated in medium (RPIJ [-1640] supplemented with 10% tallow serum). Bethoshi, 37°C Te1, 11f'l CD8, CD16, and CD20 specific Treat with target monoclonal antibody and then add rabbit complement for 1 hour at 4°C. CD1 lymphocytes were decreased by 990. Nature (London) 347: 92-95).

The obtained cell preparation mainly contains CD4 cells, B lymphocytes, and macrophages. It consisted of ji. 25 wells of NJm/well in 0.5 ml of medium. Plates were seeded with cells. Duplicate wells were filled with recombinant H[V-1 particles or Laren/U, V, or inactivated HIV-1 pillion at different concentrations (see Table ①). and Table V 4. ) was given. All wells have a final concentration of 1 μg/ml. G19.4, which reacts with T cell CD3 at a final concentration of 5%. Supplemented with 0.5 ml of medium containing Ikin 2 ([L-2) (Hoxie, J, A, et al. 1986.5science 234: 1i23-1127 ), activation of T cells by soluble CD3 MAb (Zarling, J. M. et al., 1999). 0. Nature (London> 347: 92-95).

4 or 5 days after activation (donor 129.230 or donor 231., respectively). (239), the medium was removed from the wells and the cells were incubated in PBS supplemented with 10% human serum. Washed and replaced with fresh medium. Identical samples incubated with fresh medium only (see below) or again with recombinant shell V-1 particles or inactivated HIV. -1 virus was supplemented with a corresponding concentration. As shown in Table ■, the second particle Or, at regular intervals after adding the inactivated virus, remove the culture medium from the wells. Samples were collected and the amount of p2J gagf was analyzed by specific EIA (see Section 6 above). Section 1.2). In addition, cells were simultaneously engrafted, gp120 and p24 ga HIV-1 by indirect fluorescence using a mixture of monoclonal antibodies that react with 1 protein expression was tested.

Grow recombinant particles or inactivated viruses that are washed out on day 4 or 5 The cells were fixed with anti-CD3 on day 8 to induce continuous repopulation of the cells. Transferred to new well containing MAb. On day 111, culture Samples of the supernatant were assayed for p24 gag production (Table 7). This part of the experiment However, inhibition of well production detected after treatment with recombinant HIV-1 particles is possible. This was carried out to assess whether there is a reverse phenomenon.

+3.2. result The results of the infectivity assay are shown in Tables 1 and 7. Recombinant HIV-1 capsule The offspring were isolated through culture in samples from four separate seropositive patients. inhibited the spread of virus infection. This inhibition was dose-dependent, with recombinant HrV- Equivalent to ON μg/ml of p24 gag protein in a sample treated with 1 particle Something was detected.

Busoralen/lJ, V, and inactivated viruses were also used for virus infection in culture. or in some cases (Table ■, donor Z29.6 day sample) remanufactured) (compared to IV-1 particles, IO (5 or more inactivated viruses are required) Ta.

On the 4th day, the recombinant V-1 particles or inactivated virus were washed and no longer contained. Permanent inhibition of 924 gag production by cells (Table 7) was achieved by removing particles. It also suggested that this is not enough to reverse the phenomenon. What is of interest This data also shows that silk-transformed body particles are more effective than inactivated HIV-1 pillions. (Table 7, both donors) .

(Margins below this page) Table ■ Inhibition of old V-1 virus infectivity by recombinant HIV-] particles 1, recombinant H[ The concentration of V-1 particles and inactivated virus is p24 gagl! described in relation to It is. The relative envelope glycoprotein content of these preparations is p24 ga It is 5-10% of the content of g.

Table V Inhibition of HIV infection by recombinant H[V] particles is reversible using inactivated HIV 10>44 1. The concentration of the treatment is as listed in Table ①.

Inhibition of 2.p24 gag production was assayed 111 days after activation. cells for 4 days Eyes are washed and collected in the absence of treatment. To ensure cell proliferation, cells 5%] 1-2, and on day 8 (this fixed CD3 MAb + two did.

14. Example: Recombinant IIH in vivo! V-1 particle immunity! Listed below Is the experiment non-? To evaluate the in vivo immunogenicity of recombinant V-1 particles manufactured by I. was carried out using a small animal model. Various forms of HIV-based Does any prior literature indicate that neutralizing antibodies are present in rabbits immunized with the immunogen? So rabbits were chosen for this study.

The humoral immune response of each immunized rabbit was determined by ELISA and Western Evaluation was made by ELISA measures overall H[~r-specific antibody titers. Western blot analysis allows for the analysis of individual viral proteins. to clarify the reactivity of antibodies with. In addition, the details shown in the two immunized rabbits The cellular immune response was characterized. From the results of these experiments, recombinant WHIV-1 grains It was confirmed that the offspring were capable of generating a classical Thor-specific immune response within the immunized animals.

14, 1. Immunogenicity assay Two New Sealant white female rabbits were treated with recombinant V-1 particles (Section 6 above) or were immunized with either busoralen-inactivated H[V-] virus. used immunization The amount of original protein depends on the relative p24 gag protein concentration determined by capture EIA. Previously, it was normalized (Section 6.1.2 above). immunization schedule The rules are shown in Table ■ below.

For the primary immunization (10), add the immunogen in complete Freund's adjuvant at a ratio of 11 to 11. 120gg I+24 gag and 4-6 μg gp120e The same amount of alcohol as the nv protein was administered by intramuscular injection. After 5 weeks, the rabbit has 1 Administration of the dermal route of substances prepared in incomplete Freund's adjuvant in a ratio of 1:1 (2°, boost). The amount of immunogen used for 2° immunization was 190 gp24 and 10-20 μg gp120 protein (rabbit 238 and and 239), or 100 g of p24 and 5 to IO μg of gp120 (Rabbit 24+ and 243) (see Table 3). Tertiary immunity is recombinant It was demonstrated only in rabbits immunized with prepared HIV-1 particles (Table 1).

Sera collected from animals at various time intervals were collected with either fixed, whole-destroyed virus or fixed virus. for ELISA and/or Western blot analysis using purified gp120. were assayed for anti-H[V reactivity. Each experiment was performed one week before the start of the experiment. Serum was collected from the animals and used as a preimmune control. western pro For assay, purified AV virus was added to 5DS-PAGE sample buffer. lysed and electrophoresed in an acrylamide gradient (7%-15%) slab gel. Fractionated. Fractionated viral proteins are nitroselyzed using standard techniques. Transferred to a lurose filter. The filter is then divided into similar sections and was used for Estern plot analysis. Briefly, sections were blocked at 22°C. ? After inhibition with J buffer (3% dry milk in PBS, BLOTTO) for 3 h, Each section was divided into BLOTTOO, I:51 dilutions of specific rabbit serum samples in 2% NP40. The cells were incubated overnight at 4°C. Next, wash the strip thoroughly and BL OTTO0,2% NP40 medium (7) 12' T 9:/Ha'Z quality A (I Incubation for 2 hours at 22°C with CN Lasit'7'' MILFJL 1- did. Protein bands were visualized by autoradiography. Serum is , reducing p24 gag production levels, an indicator of virus production and release. The presence of neutralizing antibodies was also tested.

The presence of antibodies that neutralize LAV-BRU isolates in the serum of immunized rabbits indicates that Detection was performed using an in vitro CEM cell infectivity assay. Serum is heat inactivated and stepwise incubation in medium (RPMI-1640 supplemented with 10% tallow serum). diluted to Equal volumes of diluted serum and 30 TCID. Mix the virus filtration of and incubated at 37° for 45 minutes. C in a 96-well microtitration dish Virus challenged into O, 1 ml culture of EM cells (2 x 104 cells) The preparation (0.1 ml) was added and incubated at 37°C without occasional mixing. did. After 1 hour of incubation, the virus-serum mixture was removed from the wells and the corresponding The diluted original rabbit serum was replaced with medium containing medium. After 5 days, culture The progress of infection in Tsuchiura can be determined by measuring the p24 gag level in Tsuchiura using εrA. monitored by.

Table ■ rabbit immunization schedule Rabbit 238.239 Week 0/1″ Immunization 5 weeks = 2° Immunization 33 weeks. 3° immunity (Rabbit 238 only) 241.243 0 weeks / 1° immunity epidemic 4 weeks 2° immunity Week 18: 3° Immunization (Rabbit 24+ only) Immunogen Rabbit Immunization 1 Recombinant HIV-1 particles 238 120 μg 190 μg 140 μg 241 120μg 100μg 120μg Inactivated old V-1 239 120μg1 90μg243 120μg100μg The primary immunogen was prepared in complete Freund's adjuvant and administered intramuscularly.

Secondary and tertiary immunizations were prepared in incomplete Freund's assortment and administered subcutaneously. It was done.

N, 2. result ! , 2.1. The antibody level measured by EL[SA is lower than 14 in rabbit 238 Pre-bleeding and 1° immunization determined by ELI34 for and 239 Represents relative antibody titers in serum samples collected at 2.5 and 7 weeks later. rabbit 23 8 (Figure 14, graph A) shows the increased immunity against HrV protein after 5 weeks. showed an infectious response, which was maintained after 2° immunization (see 7-week data; i.e. 2 weeks after boost). In contrast, rabbit 239 trial 1) (Fig. 14, graph In case B), substantial reactivity with HIV proteins was detected 2 weeks after 1° immunization. It decreased after 5 weeks. However, the remarkable boosting effect of antibody titer was observed in the 7th week. was observed in serum samples taken from rabbit 239.

An explanation for the poor boost response in rabbit 238 is that the peak immune response During this period, the animals were given 2° immunization (see Section 2). (see). On the other hand, in rabbit 239, when 2° immunization was administered, the antibody titer was completely reduced. (comparison of blood draws at 2 and 5 weeks) and therefore the boost response is more dramatic. It became something like that.

Figure 15 shows fixed total disruption wells (panels l and 2) or fixed purification envelopes. Rope glycoprotein gp120 (panels 3 and 4) Shown are the results of an ELISA assay performed in rabbit serum. shown The data are end point titers at different intervals after the second immunization. The value on the horizontal axis is blood Represents the week after the desired primary immunization. The arrow indicates the time of the secondary and tertiary test 1'). (Table ■). Rabbits immunized with recombinant torso V-1 particles ( R238 and R241) and busoralen/U, V, inert rH[V-1 Both rabbits (R239 and R243) immunized with the antigen showed a profile of antigen reactivity. was generated and correlated with the boost schedule. destroyed Antibody titers in various rabbit sera reactive with all wells were mainly p24 gag protein. This reflects the reactivity with proteins, and this reflects the reactivity of recombinant H[V] and HIV-1 It constituted approximately 90% of the protein content in both on. Total virus reactivity was about two or three orders of magnitude higher than the reactivity to gp+20, but the difference was associated with low levels (5-10%) of envelope glycoproteins in the structure.

14.2.2. Figure 16 shows neutralization of HIV-1 infectivity by rabbit antiserum. Immunization by imitation torso V-1 particles or similar old V-1, (LAV-IN, BR [J isolation) (Panel A). cormorant For overall reactivity against viral proteins, neutralizing antibody titers were correlated with the start schedule. The reported titer was 75 in p24 gag production. % inhibition.

14.2.3. Cell immunity 2 answers The cellular immune responses elicited by immunization with recombinant particles are summarized in Table Vl+ below. has been done. These results indicate that lymphocytes isolated from immunized rabbits The response to the old V-1 antigen, which is an indication of memory T cell activity necessary for mediated immunity. The results show that the cells proliferated in response to specific stimulation. Stimulation index is a recombinant torso In both rabbits immunized with V-1 particles, after the tertiary immunization, the It was. The very high stimulation index measured in rabbits 243 ('yP, Figure 15, Panel B, Figure 16).

Table ■ Response of HIV-specific T cell proliferation in immunized rabbits Recombinant particles 238 Inactivated H[V 3.0 15.0 Recombinant particles N, T, 8.4 241 Inactivated HIV 2.2 5.4 Recombinant particles N, T, N, T.

U ■ Busoralen 239 Inactivated HIV 2.6 N, B.

Inactivated recombinant particles N, T, N, B.

243 Inactivated H+V 95. ON, B.

Recombinant particles 33.0, N, B.

N, B,: Not boosted N, T,: Not tested Stimulation index ￥i11 CPM built into stimulated PBL by TdR cpm14, 2.4. antibody reactivity Western blot analysis of Figure 2 shows the results of an analysis of the reactivity of immunized rabbit serum to proteins. western pro The results of the cut analysis are shown in Figure 8. The 5th week's honest feelings from Usagi 238 The primary immune response in directed against protein (Figure 17, lane 1). rabbit 238 week 7 From serum samples, EL [SA showed no obvious boosting effect or no clear boosting effect was observed. Stanplot analysis reveals whether there is a redistribution of antibody responses among the various gag proteins. , indicating the emergence of new antibody reactivity with older proteins of larger molecular weight. (Figure 17, lane 2). In contrast, significant blockage with inactivated viruses The yeast effect (Fig. 17, lane 3) is primarily due to gag protein and This was due to increased reactivity with env gp120 protein. 1st Lanes 5.6 and 7 of Figure 7 are pools of human serum obtained from serobodic active individuals. Various concentrations of the sample (l: 100, i: +ooo and 1.+00 respectively) It exhibits a reactivity of 0(1) and serves as a control antibody.

The experiments described herein were carried out using recombinant H[V-1 particles or HIV-1 periodontal particles of the present invention. Similar to CD, it tests whether it binds to cells and enters the plasma membrane by fusion. It is designed to

+5.1. Uptake assay In this assay, transfected cells are transfected with the gene encoding the CD4 molecule. HeLa cells were used. These cells have been well characterized and has been shown to support active H[V-1 infection. Cells are 10% bovine alone on glass slides in L. Rubeco-modified Eagle medium supplemented with infant serum. Proliferated. Cells were washed with PBS/1% FC9 and incubated at 37°C at 50 μg/m+! The cells were incubated with l'I recombinant V-1 particles. Then the cells are fully Washed and fixed with 3.7% rose formaldehyde solution. Fixed cells were HI Monoclonal antibody mixture that reacts with gII+20 and p24 gag of V-1 and a 1100 dilution of 22° C. for 30 minutes. cells again Thoroughly washed, affinity-purified goat anti-mouse anti-FITC conjugated The cells were incubated with 1=50 dilutions of the body. After 30 minutes at 22°C, cells were Wash with PBS/1% FCS and place in mounding medium for examination. Ta. This sample was analyzed using a confocal laser scanning microscope to reveal intracellular staining. Mirror (Confocal Laser Scanning Microscopy) : Analyzed by CLSM).

15.2. result The assay is represented by CLS phlebotomy micrographs in Figure 18. 18th Figure A shows the positive case after incubation with recombinant barrel V-1 particles. The ff118B diagram shows HeLa CDa" cells showing light. The ff118B diagram starts from the top of the sample. The optical The same cell is shown cut into different parts. The Ia microscopic panel reveals the intracellular environment. This shows that the fluorescence increases as the temperature increases, and this indicates that the The +V-1 particles were bound, indicating that they were taken up.

Here, recombinant HIV-enriched particles, busoralen/UV-inactivated HIV-I Silk vaccinia virus expressing on and HIV-1 gag and env antigens Testing the immunogenicity of recombinant HIV-1 particles in non-human primates immunized with do. The results showed that recombinant HfV-1 particles were able to neutralize antibodies against HIV-1. have been shown to elicit cell-mediated and humoral immune responses, including

+6.1. Immunization protocol According to the following schedule, 12 macaque monkeys (Macacafasci) psoralen/UV-inactivated HIV -1 pyrion, or recombinant vaccinia expressing H[V-1gag and env antigens. Immunized with the virus.

n (n=2) -Next immunogen Secondary immunogen Boosted week/Ryorin band/Anno 】band 1, Ku G2E5/No particle/[FA 82, v-G2E5/No rgp1 60/IFA 83, particles/IFA particles/IFA 44, particles, /DE TOX particles/DETOX 45, inactivated H [V/IFA inactivated HIV/I F8 46, inactivated H[V/DETOX, inactivated HIV, DETOX 4 Immunization with live recombinant vaccinia virus is administered by skin scratching. v-G2E5 (Section 7, supra) of the Braaku forming unit of lXlO7 per animal ). Recombinant HIV-like particles and Bunoralene/UV-inactivated H[V One infusion of each pillion was performed using a p24 anti-capture assay (Genetics system). ) containing the equivalent of 200 μg of p24 when measured by Immunoplot assay. It contained about 6 μg of gl)120 as measured by assay. recombinant gp 160 is a B5C infected with a recombinant vaccinia virus expressing the same antigen. - Purified from 40 cells and expressed by a recombinant body - 1 grain. A dosage level of 6 μg per immunized animal was used. all particles or The subunit immunogen consists of detoxified monophosphoryl lipid A and bacterial cells. 12! Incomplete Freund's adjuvant (Dirco) or DE TOX (RIBI Immunobiochem). Recombinant product Immunization with particles, inactivated H[V-]pyrions, or recombinant gp160 all It was performed by intramuscular injection.

+6.2. result The immune response against HIV-1 generated by these macaque monkeys is due to the following steps: Determined by: (1) total pillion EL [total by SA (7) HIV -1 specific antibody: (2) gp120 EL [SJ +: J: LeH [V-1 x envelope-specific antibodies, (3) neutralizing antibodies by focal immunoassay, and and (4) H [lymphocyte increase in response to V-1 stimulation! Cell-mediated immunity due to 1liY response Epidemic. The results are shown in Tables ~■.

(Margins below this page) Chapter ~■Table Immune response to old V-1 generated by immunized macaque monkeys Group and Immunization Animal Antibody [Answer.

Old V-1gp120 Neut S, 1. '1, v-G2E5/particle 891 33 >25600 >1600 >400 2.189149 12800 >1600 >400 115.42, v-G2E5/gp160 89147 1600 400 25 74.389138 0 800 100 69. 73, Particles (IFA) 88079 > 51200 > 800 25 3.98 9068 6400 > 1600 25 50.04, particles (DETOX) 8 8116 800 1600 25 2.889072 0 <25 0 4. 6 5. Inactivated H[V([FA) 89150 12800 80o <25 4. 789086 3200 <25 0 43.36, inactivated old V 89151 1600 1600 25 5.7 (DETOX) 89156 50 800 0 3.9 foot tall monkeys 0 0 0 0.9a Antibody responses were in group 1 and For animals in group 2, 4 weeks after secondary immunization, i.e. at 12 weeks, for animals 3-6n. It was measured at 8 weeks.

b The lymphoproliferative response was -4X after the next immunization! It was decided that The stimulation finger is (S, 1.) 1H-thymidine c, p incorporated into HIV-1-stimulated cells.

It was measured by dividing the amount by c, p, m taken up into non-stimulated cells. .

The results shown in Table ■ show the following: (1) Recombinant shell V-1 particles are immunogenic, especially recombinant vaccinia expressing both env and gag antigens. HIV-specific when used in aviral-conditioned animals. triggers a targeted immune response.

(2) It is possible to boost previously treated animals with recombinant HIV-1 particles. boosting with an equal amount of soluble gp+6o It is likely that the recombinant V-1 particles are able to increase the effectiveness of the envelope antigen. and (3) - single immunization for subsequent and secondary immunizations. When used as a source, the recombinant HIV-enriched particles are used in large numbers of immunized macaque monkeys. In many cases, the same rats were immunized with an equal amount of inactivated HIV-1 pillions. VEL elicits H[V-specific antibody and T-cell immune responses.

17. Deposit of microorganisms The following recombinant vaccinia viruses are of the American type and are of the Russian type. Co., Ltd., MD, and was given the following deposit number:

Recombinant virus deposit number v-env5 VR21+3 v-gag2 VR2265 -G2E5 The following recombinant plasmids have been deposited and described at NRRL, Veolia, Illinois. I was given a deposit number.

Plasmid host deposit number CMHiEnv5 (1104-bl) E, coli DH5aCmHIVde lkpnAvr (Gag 2TRE) (1160-at) E, coli DH5a The present invention It is not limited to the scope of the deposited virus, because the The embodiment described above is intended as one example of an aspect of the invention, and functionally equivalent embodiments are intended as one example of an aspect of the invention. Either way is within the scope of this agreement. In fact, in addition to what is written and shown here, In addition, various modifications of the present invention will become apparent to the present text from the foregoing description and accompanying drawings. Dew. Such variations are intended to be within the scope of this paper.

Number and size of all base pairs and amino acid residues for nucleotides and peptides is an ordinary meaning, and it is also understood that the description is used as a five-point. .

The nucleotide site for the H[V-1 gene is isolated from the L4V-BRIJ isolate- Based on the genome sequence of Wain-Hob Deposit No. KO2013 (Wain-Hob son et al., 1985. Ce1l 40:9317).

A day CDEFGI-11,D -father p24-″ →-1 TSPSTSPSTSTSPS fraction number tjo-4 MAb FIG. 4b FIG, 4d B Li FIG 6 FIG.7 FfG, 8 protein FIG.9C FIG, 9D p24 FIG. 108 Kunono +! :LL’) O+%+Roro OC3+0 20 30 40 50 Weeks after immunization FIG, 15A Weeks after immunization FIG, 15th FIG, 15C FIG, 15D Number of replicates after immunization F(G, 16A FIG. 17 FIG, 18A international search report 1+hl++Il Hinoki M-^”””-”6 PCT/IIsQO10679R

Claims (86)

[Claims] 1. incorporates numerous immunogenic epitopes of the native human immunodeficiency virus, Non-replicating recombinant HIV particles consisting of structured core and envelope proteins Child. 2. The core protein consists of the human immunodeficiency virus type 1 core protein. Non-replicating recombinant HIV particles according to claim 1. 3. Claim 2, wherein the human immunodeficiency virus type 1 core protein consists of p24. Non-replicating recombinant HIV particles. 4. The core protein of human immunodeficiency virus type 1 consists of p24 and p17. 2. Non-replicating recombinant HIV particles according to claim 2. 5. The envelope protein is the human immunodeficiency virus type 1 envelope protein. 2. The non-replicating recombinant HIV particle of claim 1, which consists of a proteinaceous substance. 6. 6. The non-replicating method of claim 5, wherein the HIV-1 envelope protein consists of gp41. Recombinant HIV particles. 7. The envelope proteins of human immunodeficiency virus type 1 are gp41 and gp12. 6. The non-replicating recombinant HIV particle of claim 5, consisting of 0. 8. 6. The non-duplicating method of claim 5, wherein the HIV-1 envelope protein consists of gp160. Recombinant HIV particles. 9. Claim 8, wherein gp160 is not cleaved to generate gp120 and gp41. non-replicating recombinant HIV particles. 10. 9. The non-replicating recombinant HIV particle of claim 8, wherein gp160 is truncated. Child. 11. 10. The non-replicating recombinant HIV particle of claim 9, wherein gp160 is truncated. Child. 12. 2. The non-replicating product of claim 1, wherein the core protein consists of HIV-2 core protein. Recombinant HlV particles. 13. the envelope protein consists of HIV-2 envelope protein; The non-replicating recombinant HIV particle of claim 1. 14. Envelope proteins are present in both HIV-1 and HIV-2 envelopes. 2. The non-replicating recombinant HIV particle of claim 1, which consists of a protein. 15. Constructed, incorporating multiple immunogenic epitopes of native HIV-1 Non-replicating recombinant HIV consisting of HIV-1 core and envelope proteins -1 particle. 16. Constructed, incorporating multiple immunogenic epitopes of native HIV-2 Non-replicating recombinant HIV consisting of HIV-2 core and envelope proteins -2 particles. 17. It covers a large number of immunogenic epitopes of both native HIV-1 and native HIV-2. A non-containing protein consisting of organized HIV core and envelope proteins. Replicating recombinant HIV particles. 18. An effective amount of non-replicating recombinant H to block human immunodeficiency virus infection. an individual infected with human immunodeficiency virus, comprising administering IV particles to the individual A method of halting the progression of acquired immunodeficiency syndrome in patients. 19. An effective amount of non-replicating recombinant H to block human immunodeficiency virus infection. an individual infected with human immunodeficiency virus, comprising administering IV particles to the individual A method of halting the progression of lymph node damage in patients. 20. An effective amount of non-replicating recombinant H to block human immunodeficiency virus infection. an individual infected with human immunodeficiency virus, comprising administering IV particles to the individual How to halt the progression of AIDS-related diseases in patients. 21. An effective amount of non-replicating recombinant H to block human immunodeficiency virus infection. CD by human immunodeficiency virus consisting of treating lymphocytes with IV particles A method for reducing the infectivity of 4+ lymphocytes. 22. incorporates numerous immunogenic epitopes of naturally occurring human retroviruses, A non-replicating group consisting of an organized retroviral core and envelope proteins Replacement retrovirus particles. 23. The retrovirus core protein consists of HTLV-I core protein, 23. The non-replicating recombinant retrovirus particle of claim 22. 24. Retrovirus core protein consists of HTLV-II core protein , the non-replicating recombinant retrovirus particle of claim 22. 25. The retroviral envelope protein is the HTLV-I envelope protein. 23. The non-replicating recombinant retrovirus particle of claim 22, comprising a protein. 26. The retroviral envelope protein is the HTLV-II envelope protein. 23. The non-replicating recombinant retrovirus particle of claim 22, which consists of a protein. 27. Retroviral envelope proteins are HTLV-I and HTLV-II 23. The non-replicating recombinant retro protein of claim 22, consisting of both envelope proteins of virus particles. 28. A construct that incorporates multiple immunogenic epitopes of native HTLV-I. A non-replicating, recombinant H TLV-I particles. 29. Constructs that incorporate multiple immunogenic epitopes of native HTLV-II A non-replicating recombinant protein consisting of HTLV-II core and envelope proteins manufactured by HTLV-II particles. 30. Numerous immunogenic epitopes of both native HTLV-I and HTLV-II Consists of assembled HTLV core and envelope proteins. Non-replicating recombinant HTLV particles. 31. non-replicating recombinant HTLV-I in an amount effective to block HTLV-I infection. development in an individual infected with HTLV-I, consisting of administering HTLV-I particles to the individual. A method of halting the progression of human T-cell leukemia. 32. an amount of non-replicating recombinant cleft HTLV-I effective to block HTLV-I infection; H in an individual infected with HTLV-I, consisting of administering to the individual HTLV-I particles. A method of inhibiting the progression of TLV-I associated lymphoma. 33. an amount of non-replicating recombinant HTLV effective to block HTLV-II infection; - to an individual infected with HTlV-II, comprising administering to the individual HTLV-II particles. A method for inhibiting the progression of HTLV-II associated leukemia. 34. CD4 cell surface receptors in an amount effective to block human retrovirus infection. Treating lymphocytes with non-replicating recombinant retroviral particles that can bind to cells A method that reduces the infectivity of CD4+ lymphocytes caused by human retroviruses, consisting of Law. 35. 1. A method of forming non-replicating recombinant retroviral particles, the method comprising: Retroviral core, protease and envelope proteins in animal host cells introducing a nucleotide sequence encoding a protein; (b) Retroviral core and envelope proteins mature in mammalian host cells. Simultaneously express the texture; (c) culturing mammalian host cells; and (d) culturing non-replicating recombinant retrotransplants from the culture medium. Collecting virus particles. 36. by infecting with at least one live viral vector. Nuclei that encode the viral core, protease, and envelope proteins. 36. The method of claim 35, wherein the leotide sequence is introduced into a mammalian cell. 37. Claim 36, wherein the live viral vector consists of a recombinant vaccinia virus. the method of. 38. 37. The method of claim 36, wherein the viable viral vector comprises a recombinant retrovirus. Law. 39. By transfecting with at least one DNA vector, encodes the core, protease and envelope proteins of toroviruses 36. The method of claim 35, wherein the nucleotide sequence is introduced into a mammalian cell. 40. 1. A method of forming non-replicating recombinant HIV particles comprising: (a) a mammalian host; in the cell (i) at least one recombinant vaccinia virus carrying the HIV env gene; rus and (ii) at least one having the HIV gag and protease genes. Co-infection with recombinant vaccinia virus; (b) Mature HIV env and gag encoding in infected mammalian host cells. co-expressing gene products; (c) culturing the infected mammalian host cells; and (d) culturing non-replicating recombinants from the culture medium. A method comprising: recovering manufactured HIV particles. 41. tat, rev, vif, vpr and vpu groups in mammalian host cells. at least one recombinant vaccine carrying at least one HIV gene selected from 41. The method of claim 40, further comprising infecting with a virus. 42. 40 or 40, wherein the HIV env gene is an HIV-1 env gene 41 methods. 43. 40 or 40, wherein the HIV env gene is an HIV-2 env gene. 41 methods. 44. 40 or 40, wherein the HIV env gene is the HIV-1 gag gene. 41 methods. 45. 40 or 40, wherein the HIV env gene is the HIV-2 gag gene. 41 methods. 46. Claim that the HIV protease gene is an HIV-1 protease gene The method of item 40 or 41. 47. Claim that the HIV protease gene is an HIV-2 protease gene The method of item 40 or 41. 48. 1. A method of forming non-replicating recombinant HIV particles comprising: (a) a mammalian host; Recombinant vaccinia with HIV env, gag and protease genes in cells infecting the virus; (b) HIVen for maturation within the infected mammalian host cell; co-expressing v and gag encoded gene products; (c) culturing the infected mammalian host cells; and (d) culturing non-replicating recombinants from the culture medium. A method comprising: recovering manufactured HIV particles. 49. tat, rev, vif, vpr and vpu groups in mammalian host cells. at least one recombinant vaccine carrying at least one HIV gene selected from 49. The method of claim 48, further comprising infecting with a virus. 50. 49. or claim 48, wherein the HIV env gene is an HIV-1 env gene. 49 methods. 51. 49. or claim 48, wherein the HIV env gene is an HIV-2 env gene. 49 methods. 52. 49. or claim 48, wherein the HIV gag gene is an HIV-1 gag gene. 49 methods. 53. 49. or claim 48, wherein the HIV gag gene is an HIV-2 gag gene. 49 methods. 54. Claim that the HIV protease gene is an HIV-1 protease gene The method of item 48 or 49. 55. Claim that the HIV protease gene is an HIV-2 protease gene The method of item 48 or 49. 56. 1. A method of forming non-replicating recombinant HIV particles comprising: (a) a mammalian host; Cells were transformed into recombinant plasmids carrying HIV env, gag and protease genes. (b) mature in transfected mammalian host cells; co-expressing HIVenv and gag-encoded gene products; (c) trans culturing the infected mammalian host cells; and (d) culturing the non-replicating recombinant cells from the culture medium. A method comprising: collecting HIV particles. 57. The recombinant plasmids further include tat, rev, vif, vpr and vpu claim 56, comprising at least one other HIV gene selected from the group of the method of. 58. Mammalian host cells are classified into the groups tat, rev, vif, vpr and vpu. at least one recombinant plus containing at least one HIV gene selected from 57. The method of claim 56, further comprising transfecting with Mid. 59. Claims 56 and 57, wherein the HIV env gene is the HIV-1 env gene. Or 58 methods. 60. Claims 56 and 57, wherein the HIV env gene is the HIV-2 env gene. Or 58 methods. 61. Claims 56 and 57, wherein the HIV gag gene is an HIV-1 gag gene. Or 58 methods. 62. Claims 56 and 57, wherein the HIV gag gene is an HIV-2 gag gene. Or 58 methods. 63. Claim wherein the HIV protease gene is an HIV-protease gene. Method 56, 57 or 58. 64. Claim that the HIV protease gene is an HIV-2 protease gene The method of paragraph 56, 57 or 58. 65. A method of forming non-replicating recombinant HIV particles comprising: (a) a mammalian host; Cells are treated with (i) a recombinant plasmid carrying at least the HIV env gene and (ii) with a recombinant plasmid containing at least the HIV gag and protease genes. Transfect; (b) Mature HIV env and ga in transfected mammalian host cells. co-expressing g-encoded gene products; (c) transfected mammalian hosts; culturing the principal cells; and (d) recovering non-replicating recombinant HIV particles from the culture medium; How to become something by eating things. 66. Mammalian host cells are classified into tat, rev, vif, vpr and Vpu groups. at least one recombinant plus containing at least one HIV gene selected from 66. The method of claim 65, further comprising transfecting with Mid. 67. Claim 65 or 6, wherein the HIV env gene is an HIV-env gene. Method 6. 68. 66 or 66, wherein the HIV env gene is an HIV-2 env gene. 66 methods. 69. 65 or 65, wherein the HIV gag gene is an HIV-1 gag gene. 66 methods. 70. 65 or 65, wherein the HIV gag gene is an HIV-2 gag gene. 66 methods. 71. Claim that the HIV protease gene is the HIV-1 protease gene The method of item 65 or 66. 72. Claim that the HIV protease gene is the HIV-2 protease gene The method of item 65 or 66. 73. Claim wherein the HIV env gene encodes uncleaved gp160 protein. 40, 48, 56 or 65 methods. 74. The HIV env gene encodes a truncated gp160 protein. The method of claim 40, 48, 56 or 65. 75. A method of forming non-replicating recombinant HIV-1 particles includes: (a) BSC- 40 host cells with recombinant vaccinia virus v-env5 deposited at the ATCC. co-infecting with v-gag2; (b) culturing the infected BSC-40 cells; (c) recovering non-replicating recombinant HIV-1 particles from the culture medium; How to do it. 76. 1. A method of forming non-replicating recombinant HIV particles comprising: (a) a mammalian cell; simultaneous expression of HIVenv-encoded and HIVgag-encoded structural proteins within and (b) recovering non-replicating recombinant HIV particles from the culture medium. How to do it. 77. A method of forming non-replicating recombinant HIV-1 particles comprising: (a) BSC- 40 host cells with recombinant vaccinia virus v-G2E5 deposited at the ATCC. Infect; (b) culturing the infected BSC-40 cells; and (c) removing non-replicating recombinants from the medium. A method comprising: recovering manufactured HIV-1 particles. 78. A method of forming non-replicating recombinant HIV-1 particles comprising: (a) BSC- 40 host cells with (i) recombinant vaccinia virus v-ED2 and (ii) recombinant vaccine. Co-infected with cuccinia virus v-gag2; (b) infected BSC-40 culturing the cells; and (c) recovering non-replicating recombinant HIV-1 particles from the culture medium. ; a method comprising; 79. A method of forming non-replicating recombinant HIV-1 particles includes: (a) BSC- 40 host cells with (i) recombinant vaccinia virus v-ENV5DCT and (ii) co-infecting with recombinant vaccinia virus v-gag2; (b) culturing the infected BSC-40 cells; and (c) removing non-replicating recombinants from the medium. collecting the artificially produced HIV-1 particles. 80. A method of forming non-replicating recombinant HIV-1 particles includes: (a) BSC- 40 host cells with (i) recombinant vaccinia virus v-160NC and (ii) recombinant co-infected with Evaccinia virus v-gag2; (b) infected BSC- 40 cells: and (c) circulating non-replicating, recombinantly produced HIV-1 particles from the medium. A method comprising; 81. A method of forming non-replicating recombinant HIV-1 particles includes: (a) BSC- 40 host cells with (i) recombinant vaccinia virus v-11K16ONC and (ii ) Co-infection with recombinant vaccinia virus, v-gag2; (b) culturing the infected BSC-40 cells; and (c) removing non-replicating recombinants from the medium. A method comprising: recovering manufactured HIV-1 particles. 82. A method of forming non-replicating recombinant HIV-1 particles comprising: (a) a mammal; Cells were transferred to the recombinant plasmid CmHIVdelKpnAvr ( Gag2TRE) (1160-al) and CmHiEnv5 (1104-bl). co-transfected; (b) Mature HIVenv and gag proteins in transfected CHO cells. co-expressing transfected gene products; (c) culturing transfected CHO cells and (d) recovering non-replicating recombinant HIV-1 particles from the culture medium. How to do it. 83. 1. A method of forming non-replicating recombinant HIV-1 particles comprising: (a) a mammal; Cells were transferred to the recombinant plasmid CmHIVdelKpnAvr ( Gag2TRE) (1160-al); (b) transfection. mature HIV env and gag-encoded gene products in infected CHO cells. co-expressing; (c) culturing the transfected CHO cells; and (d) A method comprising: recovering non-replicating recombinant HIV-1 particles from a culture medium. 84. 1. A method of forming non-replicating recombinant HIV-1 particles comprising: (a) a mammal; Cells were transferred to the recombinant plasmid CmHiGag2Rre (115 Co-transfection with CmHIVdelXmn(1133-al) and CmHIVdelXmn(1133-al) Toshi; (b) Mature HIVenv and gag proteins in transfected CHO cells. co-expressing transfected gene products; (c) culturing transfected CHO cells and (d) recovering non-replicating recombinant HIV-1 particles from the culture medium. How to do it. 85. 1. A method of forming non-replicating recombinant HIV-1 particles comprising: (a) a mammal; Cells were transferred to the recombinant plasmid CmHIVdelXmn (113 Co-transfected with CmvGag2Rre (1159-al) and CmvGag2Rre (1159-al) death; (b) Mature HIVenv and gag proteins in transfected CHO cells. co-expressing transfected gene products; (c) culturing transfected CHO cells and (d) recovering non-replicating recombinant HIV-1 particles from the culture medium. How to do it. 86. 1. A method of forming non-replicating recombinant HIV-1 particles comprising: (a) a mammal; Recombinant plasmids CmHiGag2Rre, CmH deposited at NRRL co-transfected with iEnv5, CmHiTat and CmHiRev; (b) Mature HIVenv and gag proteins in transfected CHO cells. co-expressing transfected gene products; (c) culturing transfected CHO cells and (d) recovering non-replicating recombinant HIV-1 particles from the culture medium. How to do it.