EP0502105A1 - Particules retrovirales non replicantes produites par recombinaton employees comme agents antiviraux et immunogenes - Google Patents

Particules retrovirales non replicantes produites par recombinaton employees comme agents antiviraux et immunogenes

Info

Publication number
EP0502105A1
EP0502105A1 EP19910900526 EP91900526A EP0502105A1 EP 0502105 A1 EP0502105 A1 EP 0502105A1 EP 19910900526 EP19910900526 EP 19910900526 EP 91900526 A EP91900526 A EP 91900526A EP 0502105 A1 EP0502105 A1 EP 0502105A1
Authority
EP
European Patent Office
Prior art keywords
hiv
recombinant
particles
gene
nonreplicating
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19910900526
Other languages
German (de)
English (en)
Other versions
EP0502105A4 (en
Inventor
Omar K. Haffar
Shiu-Lok Hu
Allen W. Senear
Bruce M. Travis
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oncogen LP
Original Assignee
Oncogen LP
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Oncogen LP filed Critical Oncogen LP
Publication of EP0502105A1 publication Critical patent/EP0502105A1/fr
Publication of EP0502105A4 publication Critical patent/EP0502105A4/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention is directed to noninfectious recombinant-made retroviral particles, to in vitro systems by which such particles can be generated, and to their use as anti-viral agents and as immunogens for prophylaxis and therapy against human retroviruses such as the human immunodeficiency virus (HIV) .
  • Recombinant-made HIV particles of the invention incorporate correctly processed HIV core and envelope proteins and are morphologically and immunologically very similar to native HIV. Yet, since the recombinant-made HIV particles of the invention do not contain all elements of the HIV genome necessary for viral replication, they are non-infectious.
  • human retroviruses Two types have been identified, leukemia viruses and AIDS or AIDS-related viruses.
  • the primary targets of the human retroviruses are T lymphocytes and cells of the central nervous system. All human retroviruses are transmitted by intimate contact, blood contamination, and infection in utero or after birth by milk. It is likely that all human retroviruses orginated in Africa and that they encountered the human species via interspecies infection, possibly from African Green Monkeys or a related species.
  • HTLV-I Human T Lymphotropic Virus Type 1
  • HTLV-II Human T Lymphotropic Virus Type II
  • HAV Human Immunodeficiency Viruses
  • HTLV-II 5 and HTLV-II are stable; and (2) HIV entered human populations much more recently than HTLV-I or HTLV-II.
  • HIV human immunodeficiency virus
  • LAV lymphadenopathy-associated virus
  • HTLV-III human T lymphotropic virus type III
  • SIV simian immunodeficiency virus
  • HIV is a member of the nontransforming, cytopathic lentivirus family of retroviruses. HIV causes a typically fatal disease characterized by severe immunodeficiency or neurodegenerative disease, or both.
  • the primary basis for HIV induced immunosuppression is the depletion of the helper/inducer subset of T lymphocytes expressing the CD4 molecule (T4 or CD4 cells) , which serves as the high affinity cell surface receptor for the virus.
  • T4 lymphocytes are involved directly or indirectly in the induction of nearly every immunologic function in the body, and their depletion results in susceptibility to a wide range of opportunistic infections and neoplasms.
  • NK functional natural killer
  • CD4 cells + . . .
  • Infection of CD4 cells is initiated by the interaction of the CD4 molecule with the major HIV envelope glycoprotein gpl20, an event which is followed by internalization and uncoating of the virion, transcription of genomic RNA to DNA by virus-encoded reverse transcriptase, and integration of the resulting proviral DNA into host cell chromosomal DNA.
  • unintegrated proviral DNA accumulates in large amounts within infected cells and is probably a significant factor in HIV cytopathicity (Shaw et al., 1984, Science 226: 1165).
  • mRNA transcripts of integrated proviral DNA are translated into HIV proteins. These proteins are then processed and assembled along with HIV genomic RNA. Mature virions bud from the surface of infected T- 5 lymphocytes and bud internally in macrophages, incorporating host cell membrane lipid to form virion envelope.
  • HIV exerts its cytopathic effect is unknown, though several mechanisms have been proposed (e.g., accumulation of large amounts of unintegrated viral DNA in infected cells; increase in cell membrane permeability when large amounts of virus bud from the cell surface; speculations that HIV may
  • monocytes and macrophages play a major 5 role in the pathogenesis of HIV infection is compelling.
  • some subsets of monocyte-macrophages express the CD4 surface antigen and are therefore capable of binding to the HIV envelope.
  • the monocyte-macrophage is the primary cell type
  • HIV can survive in a dormant state within the monocyte-macrophag .
  • Infected mono ⁇ ytes do not exhibit the cytolytic effect that HIV has on T4 cells, perhaps due to a lower density of CD4 cell surface receptors.
  • Monocytes can therefore serve as HIV reservoirs which may ultimately transport the virus to the brain, central nervous system, and various organs in the body. It is likely that the virus crosses the blood/brain barrier within monocytes where it affects the release of monokines, enzymes, and chemotactic factors resulting in the destruction or damage of neurons and inflammation of brain tissue (Ho et al., 1987, N. Engl. J. Med. 311: 278; Fauci, 1988, Science 239: 617).
  • HIV virion is a spherical particle of about 100 to 120 nm across and contains an electron dense, tubular core comprised of the p24 gag protein, a submembrane matrix comprised of gag pl7, and an envelope comprised of the env proteins gpl20 and gp41 interspersed within the lipid bilayer membrane.
  • HIV genomic RNA is housed within the core as part of the ribonuclear protein (RNP) complex which incorporates reverse transcriptase molecules (the enzyme which catalyzes transcription of RNA to proviral DNA) and core proteins.
  • RNP ribonuclear protein
  • the protrusions comprise gpl20, which is loosely connected to its transmembrane gp41 anchor. Envelope gpl20 is spontaneously shed to a high degree from the surface of the virus, a phenomenon which may influence HIV pathogenicity. Virus particle maturation takes place both during and just after the budding process. After budding from the surface of the infected cell, HIV core proteins are cleaved from precursors by an HIV-encoded protease into mature structural proteins which organize to form the core structure.
  • HIV genome contains three genes that encode the major structural components of the virion: env (which codes for the envelope proteins) , gag (which codes for the core proteins) , and pol (which codes for reverse transcriptase, protease, and endonuclease enzymes) . These three genes are flanked by stretches of nucleotides called long terminal repeats (LTRs) .
  • LTRs include sequences that have a role in controlling the expression of viral genes.
  • the genome of HIV includes at least six additional genes, three of which have known regulatory functions. Expression of these regulatory genes is thought to have an impact on HIV pathogenesis.
  • the tat gene encodes a protein that functions as a potent trans- activator of HIV gene expression and, therefore, plays an important role in the amplification of virus replication.
  • the rev gene product regulates the splicing and transport of HIV mRNA.
  • the nef gene may down regulate virus expression.
  • the vif gene is not absolutely required for virion formation, but is critical to the efficient generation of infectious virions and influences virus transmission in vitro.
  • the Vpr gene encodes an immunogenic protein of unknown function.
  • the recently described Vpu open reading frame encodes a protein involved in the regulation of virus maturation and cytopathic effect.
  • HIV-1 variants some of which are antigenically diverse, have been isolated from individual AIDS patients over the course of an infection. Isolates may differ with respect to their tropism for specific cell types. In this regard, certain isolates appear to replicate preferentially in either CD4 + T cells or in brain-derived macrophages, suggesting that HIV infection results in different clinical manifestations due to a selective pathogenicity mechanism.
  • the immunogenic property of a given epitope such as the group-specific neutralizing epitope(s) of adenovirus hexon, may be conformation-dependent.
  • the inclusion of core antigen as an immunogen may elicit broadly reactive immune responses to different HIV-1 isolates since the core antigens of HIV are relatively conserved among various isolates. Therefore, it is of interest and importance to design and evaluate vaccines that combine the advantages of both traditional and recombinant approaches, i.e., recombinant-made vaccines that preserve the immunogenic properties of native virions yet lack the infectivity and other potential disadvantages of whole virus preparations.
  • vaccines may also be useful for post-exposure immunotherapy.
  • current rabies vaccines are given to individuals following potential exposure to rabies viruses. It has been proposed that immunotherapy could also be of value in preventing AIDS in HIV infected individuals, since there is a long period of c latency between infection and disease progression (Salk, 1987, Nature 327:473-476).
  • the present invention is directed to nonreplicating 0 recombinant-made retroviral particles, vaccine formulations comprising nonreplicating recombinant-made retroviral particles, methods for the generation of nonreplicating recombinant-made retroviral particles, and the use of nonreplicating recombinant-made retroviral particles as 5 antiviral agents.
  • the recombinant-made retroviral particles of the invention comprise retroviral core and envelope proteins assembled into structures having immunological and morphological characteristics that closely resemble those of native retrovirus virions.
  • the primary structural 0 difference between the recombinant-made retroviral particles of the invention and native retroviral particles is the absence of a complete retroviral genome in the former.
  • the recombinant-made retroviral particles of the invention are totally noninfectious and can not reproduce. Yet, because the recombinant-made retroviral particles are structurally organized as are infectious retroviral particles, they are 0 highly immunogenic and are capable not only of eliciting a protective immune response against the particular retrovirus of interest, but are also effective at blocking retrovirus infectivity.
  • Applicants' method for generating the nonreplicating 5 recombinant-made retroviral particles of the invention involves the coexpression of retroviral core and envelope structural proteins in mammalian host cells capable of directing their maturation and supporting their association c into correctly assembled budding particles.
  • Introduction of the nucleotide sequences encoding such retroviral core and envelope structural proteins into the mammalian host cell • may be accomplished using several established techniques such as, for example, infection by live virus vectors and Q transfection with DNA vectors.
  • a nucleotide sequence encoding retroviral protease should also be introduced into the mammalian host cell in order to ensure the proper processing of the retroviral core proteins.
  • the present invention is directed to nonreplicating recombinant-made HIV particles, vaccines against Human Immunodeficiency Virus, methods for generating nonreplicating recombinant-made HIV particles, and the use of nonreplicating recombinant-made HIV particles to inhibit o HIV infection and to treat individuals infected with HIV.
  • recombinant vaccinia viruses are used as vectors to introduce the gag, protease and envelope genes of Human Immunodeficiency Virus into mammalian host cells which 5 direct the generation of HIV-1-like particles having immunological and morphological characteristics closely resembling those of native HIV-1.
  • These recombinant-made HIV-l particles are able to block the infectivity of live HIV in vitro and are highly immunogenic in vivo.
  • FIG. 1 Radioimmunoprecipitation analysis of HIV-l proteins expressed in BSC-40 cells infected with recombinant vaccinia viruses. Monolayers of BSC-40 cells were grown to 5 confluency in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal calf serum (FCS) .
  • DMEM Dulbecco's Modified Eagles Medium
  • FCS fetal calf serum
  • v-env5 (lanes A and B) , v-env5 + v-gag2 (lanes C and D) , v-gag2 (lanes E and F) , or v-NY parental 5 virus (lanes G and H) at a MOI of 10 PFU/cell of each virus.
  • the cells were radiolabeled for 4 hours with [ 3 S]-methionine and [ S]-cysteine (100 uCi/ml) .
  • Culture media was collected and the cells washed with PBS, harvested, and lysed in RIP buffer (1% NP40, 0.5%
  • FIG. 2 Cell surface compartmentalization of the HIV envelope proteins synthesized by infected BSC-40 cells.
  • FIG. 3 Isolation of recombinant HIV particles
  • BSC-40 cells infected as described in FIG. 2 were radiolabeled at 5 hours post-infection with [ 35S]-methionine and [ 35S]-cyste ⁇ ne (60 ⁇ Ci/ml) for 10 hours.
  • the culture media from each infection condition (14 ml) was collected
  • the resulting particulate pellet (P) was resuspended in RIP buffer.
  • the P fractions (lanes B, E, and H) were assayed for HIV proteins in parallel with 2 ml (from 12 ml total) S fractions (lanes C, F, and I) and the 2 ml TS material (lanes A, D, and G) by RIP as described in Section 6.1.2., infra.
  • the gradient fractions were collected from the bottom of the gradient in 200 ul aliquots.
  • the collected material was halved and assayed for gag p24 content by EIA as described in Section 6.1.2., infra.
  • the peak fractions of the EIA are presented as ng of p24 detected per fraction collected (closed circles) , and can be correlated to sucrose concentration (open circles) .
  • the fractions constituting the top of the gradient did not contain any p24 as confirmed by western blot analysis.
  • FIG. 4 Analysis of assembled recombinant HIV-1 particles by thin section electron microscopy and immuno electron microscopy.
  • MAbs 110-4 or 41-1 as ascites fluid (1:2000 in blocking buffer) were added to the various samples as indicated.
  • FIG. 5 Nucleic acid content of recombinant-made HIV-1 particles, determined by dot blot hybridization assay as described in Section 6.2.4., infra. Panel A: gag-specific probe. Panel B: env-specific probe. Recombinant-made HIV-l particle and inactivated HIV virion concentrations were determined as ng p24 equivalents.
  • Panel C line drawing indicating the coordinates for the gag-pol gene (258-3317) and the env gene (5671-8572) used in the preparation of the recombinant vaccinia viruses v-gag2 and v-env5, respectively.
  • the arrow indicates the position of the RNA packaging sequence (300- 319) located upstream of the gag-pol gene (Lever et al., 1989, J. Virol. 63:4085-4087).
  • FIG. 6 Schematic representation of the construction of plasmid pv-G2E5.
  • FIG. 7 Western Blot of Recombinant Vaccinia Virus Infected Cells.
  • BSC-40 cells were infected at an MOI of 5 pfu/cell and harvested for PAGE at 24 hours post infection.
  • Infected cell lysates were electrophoresed in a 7-15% aerylimide-gel and electro-transferred to nitrocellulose.
  • Immunoblots were reacted with HIV + Human Serum (Trimar) and then peroxidase conjugated goat-anti-human IgG. Blots were visualized using 2-Chloro-Napthol as substrate. Gel lanes were loaded as indicated.
  • FIG. 8 Radioimmunoprecipitation analysis of recombinant-made HIV-1 particles produced by V-G2E5 infected BSC-40 cells .
  • FIG. 9 Schematic diagrams of plasmid vector constructs described in Section 8.1., infra.
  • A CmHIVdelXmn(ll33-al) and CmHIVdelKpnAvr(Gag2TRE) (1160-al) .
  • B CmHiGag2Rre(1158- al) and CmvGag2Rre(1159-al) .
  • C CmHiEnv5(1104-bl) and
  • FIG. 10 Immunoreactivity of recombinant-made HIV-1 particles generated in transfected CHO cells.
  • Recombinant- made HIV Particles were collected from the culture medium of 3010-C6 cells, concentrated by high speed centrigution and fractionated by banding in a 15-60% sucrose gradient.
  • Bottom Aliquots of selected fractions (indicated on top panel by *) were analyzed by electrophoresis on a 7- 15% gradient selected fractions polyacrylamide gel and electro-transfer to a nitrocellulose filter which was probed with a human AIDS patient serum and 125-1 labeled Protein A. Also loaded were an aliquot of unfractionated particles and isolated HIV virus.
  • FIG. 11 Analysis of HIV-specific antigens expressed in HeLa cells transfected with plasmid vectors encoding HIV-1 env, tat and rev genes.
  • HeLa cells were contransfected with CmHiTgfbEnv ⁇ (each lane) plus the tat and rev plasmids indicated above each lane ("Bs" is a control plasmid comprised of the "Bluescribe plus" vector with no coding sequences inserted) .
  • Zinc was added to cultures, indicated by * f +Zn', 24 hours before samples were collected.
  • BSC-40 cells were infected at an MOI of 10 pfu/cell and harvested for PAGE at 23 hours post infection. Infected cell lysated were electrophoresed in a 8.5% acrylimide gel and electro-transferred to nitrocellulose. Immunoblots were reacted with HIV+ Human Serum (Trimar) and then peroxidase conjugated goat-anti-human IgG. Blots were visualized using 2-Chloro-Napthol as substrate. Gel lanes were loaded as indicated.
  • FIG. 13 Analysis of the infectivity of T-lymphoblastoid cells with recombinant-made HIV-1 particles.
  • T lymphoblastoid cells CEM
  • CEM T lymphoblastoid cells
  • Five days post infection cell samples were collected from each well and analyzed for intracellular HIV antigens by indirect immunofluoresence, as described in Section 7.1, infra. Evans Blue dye (red stain) was used to facilitate localization of cells.
  • Panel A CEM cells incubated with recombinant-made HIV-1 particles.
  • Panel B CEM cells incubated with HIV virus, showing positive fluorescence.
  • Panel C Syncytium of CEM cells incubated with HIV virus.
  • FIG. 15 Humoral immune response in immunized animals. New Zealand white rabbits were immunized with recombinant- made HIV-1 particles (R#238 and R#241) and inactivated HIV-1 virions (R#239 and R#243) . At different intervals following the primary immunizations serum samples were collected and assayed for HIV-1 specific antibodies by ELISA on disrupted whole virus (panels A and B) or on purified gpl20 (panels C and D) . The data presented (ordinate) are the end point titers of HIV-1 specific antibodies calculated at 2 fold the preimmune serum titers. The abscissa values represent the schedule in weeks post-primary immunizations when serum samples were collected. The arrows indicate the times of the secondary (wk4 R#241 and 243, wk5 R#238 and 239) and tertiary (wkl8 R#241, wk33 #R238) immunizations.
  • FIG. 16 Neutralization of HIV-1 infectivity of CEM cells with the rabbit sera.
  • Selected serum samples from the immunized rabbits (panel A R#238 and R#241, panel B R#239 and R#243) were assayed for HIV-1 specific neutralizing activity.
  • the homologous virus (BRU isolate) was preincubated with the appropriate sera for 45 minutes at 37'C prior to addition to the cells. After 1 hour at 37° the virus and sera were removed from the cells and replaced with appropriate dilution of sera in culture medium.
  • Neutralization was determined by measuring, using a EIA, the reduction in p24 ⁇ a ⁇ protein released from the cells. The reported neutralizing titers (ordinate) were for 75% reduction in p24 levels in the culture medium. The abscissa values are as described in the description of FIG. 15.
  • FIG. 17. Antibody reactivity with individual viral proteins as determined by Western blot analysis. Details of the experimental procedure used and discussion of the results are presented in Section 14.2.4., infra.
  • FIG. 18 Confocal Laser Scanning Micrographs of HeLa cells transfected with CD4 gene incubated with recombinant- made HIV-1 particles then with HIV specific antibodies. Details of the procedure are set forth in Section 15.1., infra. 5. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention relates to nonreplicating recombinant-made retroviral particles which closely resemble live retrovirus virions in their immunological, structural, and morphological features.
  • the method of the invention is applicable to the construction of recombinant-made particles resembling any of the human retroviruses (e.g., HTLV-I, HTLV-II, HIV-1, HIV-2).
  • a particular aspect of the invention relates to recombinant-made HIV particles.
  • Recombinant-made HIV particles like native HIV, are comprised of correctly processed and assembled HIV core and envelope proteins which retain immunoreactivity to anti-HIV sera.
  • the recombinant- made HIV particles do not, however, contain HIV genome and are therefore nonreplicating.
  • the method of the invention is illustrated by examples in which a novel in vitro system is used to generate recombinant-made HIV-1 particles displaying gpl20/gp41 envelope protein complexes on their surfaces.
  • the recombinant-made HIV particles closely resemble authentic HIV virions, both in morphology and in antigenic properties. When used as an immunogen, these particles will present antigens in a manner similar to presentation of antigen during HIV infection, thereby eliciting immune responses that are highly relevant and potentially protective against natural infection. This feature of the invention can not be achieved by any recombinant subunit vaccine described to date.
  • the approach of the invention being based on recombinant .DNA techniques, provides flexibility for incorporating antigens from diverse isolates of HIV-l and HIV-2, thereby generating cross-reactive immune responses considered essential for an effective vaccine against AIDS.
  • Recombinant DNA techniques may also be used to delete or modify potentially harmful epitopes that contribute to any enhanced infectivity or pathogenicity of the virus.
  • the recombinant-made HIV particles described herein are not infectious and do not contain complete HIV genome. Therefore, immunization with these particles does not introduce the risk of infection potentially associated with inactivated or attenuated whole virus vaccines.
  • recombinant-made HIV particles of the invention include their use as an anti ⁇ viral agents which interfere with HIV infection, their use in raising monoclonal antibodies to HIV core and envelope protein antigens, their use in the development of anti- idiotypic antibodies, and their use in elucidating the process of HIV encapsidation.
  • the recombinant-made HIV-l particles of the invention demonstrate antiviral effect (Sections 12 and 13, infra) and elicit HIV-specific humoral and cellular immune responses in both rabbits and macaque monkeys immunized with the particles (Sections 14 and 16, respectively) .
  • Applicants' method for generating recombinant-made HIV particles involves the coexpression of the HIV env-encoded and gag-encoded structural proteins in mammalian cells.
  • the cultured host cells of choice must be capable of synthesizing and correctly processing HIV proteins.
  • Introduction of the env and gag genes into host cells may be accomplished using a variety of established techniques known in the art including infection by live virus vectors, such as vaccinia virus and retroviral vectors, and transfection using DNA vectors.
  • the recombinant-made HIV particles may be isolated from the culture media using techniques standard in the art.
  • infra recombinant-made HIV particles are produced in African green monkey kidney (BSC-40) cells coinfected with two recombinant vaccinia viruses, one carrying the complete gag gene and the other carrying the complete env gene of HIV-1.
  • BSC-40 African green monkey kidney
  • This double infection results in the budding of assembled, recombinant- made HIV-1 particles from the surface of the BSC-40 cells.
  • Biochemical analysis revealed that these particles incorporated mature, immunoreactive gag and env proteins.
  • the morphology of the recombinant-made particles, as visualized by electron microscopy, is virtually the same as live HIV.
  • an alternative system for generating the recombinant retroviral particles of the invention using viral vectors is demonstrated by way of examples in which a single recombinant vaccinia virus containing both the env and gag genes of HIV-1 is used to transfect mammalian cells which then generate recombinant- made HIV-1 particles (Section 7, et seq., infra) .
  • recombinant-made retroviral particles may be generated using a system involving mammalian cells transfected with DNA encoding the retroviral structural proteins.
  • a system involving mammalian cells transfected with DNA encoding the retroviral structural proteins As one example of this embodiment, described in further detail in Section 8, et seq., infra, two plasmid vectors encoding, respectively, the HIV-1 gag 5 and HIV-1 env genes, are used to transfect CHO cells, which then direct the synthesis of HIV-1 gag and env antigens assembled into recombinant-made HIV-1 particles.
  • Recombinant DNA vectors and viral vectors such as vaccinia viruses may be constructed according to the methods outlined in copending United States Patent Applications Serial No. 779,909 filed September 25, 1985; Serial No.
  • recombinant vaccinia viruses carrying HIV env and gag sequences are constructed and used as vectors. Briefly, plasmid vectors containing HIV core and envelope protein coding sequences under the transcriptional control of the vaccinia promoter are constructed and used to affect the integration of the HIV gene sequences into the vaccinia virus genome by vivo recombination. Recombinant vaccinia viruses are identified, purified, and evaluated for their ability to direct the synthesis of HIV proteins in infected cells, as described in the above-referenced copending patent applications.
  • recombinant plasmid vectors encoding various combinations of HIV structural and/or regulatory genes are constructed and used as vectors for transfecting cells capable of generating recombinant-made HIV particles.
  • the constructions of a representative range of such vectors are described in Section 8., et seq., infra.
  • the precise nature of the individual protein components of the recombinant-made particles of the invention may be modified by recombinant DNA techniques, during construction of the recombinant DNA vectors, recombinant vaccinia virus vectors, etc. In this way, the existence and structural composition of retroviral epitopes presented on the particles may be defined.
  • variable epitopes of HIV gpl20 from the different HIV isolates may be included to generate a cross-reactive immune response.
  • different HIV gag gene sequences may be incorporated within recombinant vectors to vary the immunogenicity of recombinant-made HIV particles.
  • Vectors encoding mutated HIV gene sequences may also be useful in generating recombinant-made HIV particles, which may result improved in immunogenicity, anti-viral effect, etc.
  • recombinant-made retroviral particles is controlled to a large extent not only by the composition of the recombinant vectors used but also by the combination of vectors used in the infection or transfection process, this choice being a primary variable in the overall method of the invention.
  • infecting BSC-40 host cells with a single recombinant vaccinia strain carrying the HIV-1 gag gene (v- gag2) resulted in the formation of HIV-l core proteins assembled into particles.
  • host cells may be transfected with a single plasmid vector encoding both HIV env and gag, or a vector encoding HIV env, gag and other HIV genes, etc.
  • Cells may also be transfected with a plurality of plasmid vectors, each encoding a different HIV gene or combinations of HIV genes.
  • transfected cell lines may be transfected again with vectors designed to add the expression of other HIV genes to the particle generation system.
  • Vectors encoding regulatable promoters may be used to modulate the expression of HIV proteins in transfected host cells (see, for example, Section 8.3.2., infra) .
  • Vectors encoding other HIV genes may be used to affect their expression in conjunction with the expression of HIV structural genes in transfected cells. The expression of such HIV regulatory and/or accessory proteins in the system may be used as a means of altering particle characteristics and/or their production levels.
  • HIV protease is not required for the assembly of core particles, its role in the formation of infectious virion is indicated (Peng et al., 1989, Virology 63: 2550). As such, it may be advantageous to include HIV protease function in the production of recombinant-made HIV particles such that they may closely resemble native virions.
  • gag genes from HIV-1, HIV-2, or different isolates thereof may be used to construct recombinant vectors.
  • the multi-infection approach illustrated above, and similar approaches using other recombinant vectors may be used to generate recombinant HIV particles having a broad range of surface and core antigen characteristics.
  • the number of different combinations are essentially unlimited.
  • the particular host cell selected will also influence the nature of the particles produced by the method of the invention. Cells should be chosen for their ability to express and correctly process mature HIV proteins. Since the HIV envelope proteins gpl20 and gp41 are glycosylated and are derived by proteolytic cleavage from a larger gpl60 precursor, a cell capable of directing these post- translational processing modifications is desirable.
  • the host cell must be susceptible to infection with recombinant virus.
  • host cells of human, simian, or rodent origin are used.
  • Host cells may be infected by recombinant vaccinia virus according to the conditions described in Section 6., infra, or transfected by recombinant plasmid vectors as described in Section 8., et seq., infra.
  • cells When utilizing a recombinant vaccinia vector system, such as that described in Section 6., et seq., infra, cells may be infected at a multiplicity of infection (MOI) of about 10 PFU per cell of each recombinant vaccinia virus.
  • MOI multiplicity of infection
  • a multiplicity of infection such as that described in Section 6.
  • MOI multiplicity of infection
  • increasing or decreasing the MOI for one or more recombinant vaccinia may be used to influence the nature of the resulting particles. This would be an important factor in designing polytropic or heterologous particles. For example, a desired ratio of HIV-1 to HIV-2 envelope antigens on the surface of a heterologous particle may
  • infra recombinant vaccinia viruses v-env5 and v-gag2 were used to coinfect cultured African green monkey kidney (BSC-40) cells.
  • BSC-40 African green monkey kidney
  • the infected BSC-40 cells synthesized HIV-1 envelope proteins gpl20, gp41, and the gpl60 precursor, as well as HIV-1 gag proteins p24, pl7, pl5, p55, p45, and p39.
  • At least p24, pl7, gpl20 and gp41 assemble particles that are immunoreactive to polyclonal anti-HIV-1 sera and monoclonal antibodies specific for pl7, p24, gpl20 and gp41.
  • Ultrastructural analysis of the recombinant-made HIV-1 particles by thin section electron microscopy and immunogold labeling revealed substantial morphologically identity with native HIV.
  • recombinant-made HIV-1 particles were visualized as spherical objects having a diameter of between 100 and 120 nm, and contained an electron dense inner core which was either rod-shaped or spherical, depending on the section angle.
  • Various forms of immature particles were also visualized, consistent with the course of HIV-l virion morphogenesis.
  • the recombinant-made HIV particles of the invention may also be generated by cells transfected with recombinant plasmid vectors encoding HIV gag, env, and other HIV genes.
  • recombinant plasmid vectors encoding HIV gag, env, and other HIV genes.
  • Various specific embodiments of this aspect of the invention are described in Section 8.2. infra.
  • Chinese Hamster Ovary (CHO) cells are transfected with plasmid vectors encoding HIV-1 gag, env, tat, and rev genes.
  • Stable CHO cell lines expressing and secreting processed env and gag proteins as recombinant-made HIV-1 particles are obtained. Similar results may be obtained using HeLa, BSC-40 and Vero cells transfected with various plasmids or combinations thereof (See, for example, Sections 8.3.1., 8.3.2., and 8.3.3., infra).
  • Recombinant-made particles may be identified by a variety of immunochemical means and/or by electron microscopy (EM) visualization.
  • Immunochemical detection methods such as radioimmunoprecipitation (RIP) , capture enzyme immunoassay (EIA) , western blot analysis and the like may be used. Specific examples of how these techniques may be used to identify recombinant-made HIV particles are described by way of example in Section 6., infra.
  • Various EM techniques may be used to identify and characterize recombinant-made HIV particles, such as the thin section EM and immuno electron microscopy techniques described in Section 6.1.3., infra.
  • SEM scanning electron microscopy
  • surface replica electron microscopy elucidation of HIV fine structure
  • Recombinant-made particles may be isolated from the culture media of host cells using standard techniques well known in the art. It is important, however, to use isolation methods which minimize the degree of gpl20 shedding in order to maximize the immunogenicity of recombinant-made HIV particles. 5.3. DETERMINATION OF THE IMMUNOGENICITY OF
  • the immunogenicity of the recombinant-made particles can be determined by monitoring the the immune response of test animals following immunization.
  • Test animals may include mice, rabbits, chimpanzees, and eventually humans.
  • routes of immunization may be considered, including oral, intrader al, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, etc.
  • the immune response induced by recombinant-made HIV particle immunogens can be analyzed by three approaches: (a) the reactivity of the resultant immune sera to authentic HIV antigens using known techniques such as enzyme linked immunosorbent assay (ELISA) , im unoblot, radioimmunoprecipitation, etc.
  • ELISA enzyme linked immunosorbent assay
  • the immunogenicity of the recombinant-made HIV-1 particles is evaluated in non-human primates (Section 16., et seq., infra). More particularly, macaque monkeys are immunized with recombinant-made HIV-l particles in conjunction with other HIV-1 antigens. Immune responses in immunized subjects may be determined by various assays, including whole virion ELISA, gpl20 ELISA, focal immunoassay and lymphoproliferative response assay.
  • recombinant-made HIV-l particles when used as the sole immunogen for primary and secondary immunizations, recombinant-made HIV-l particles elicit HIV-specific humoral and cellular immune responses.
  • the recombinant-made HIV-1 particles were particularly effective when used to immunize animals previously primed with a recombinant vaccinia virus encoding HIV-1 env and gag antigens.
  • a specific embodiment of the invention is the formulation of vaccines capable of invoking immune responses that contribute to the prevention of retrovirus infection or the development of retrovirus-associated diseases such as AIDS.
  • the vaccine formulations use the recombinant-made retroviral particles of the invention as immunogens which, by combining major retroviral core and envelope proteins, are multivalent in nature.
  • the recombinant-made retroviral particles may be used as specific immunologic enhancers that may be used to ameliorate the progression of retrovirus-associated diseases in persons already infected with retrovirus.
  • a further particular embodiment of the invention involves vaccine formulations capable of invoking immune responses that contribute to the prevention of HIV infection or the development of AIDS.
  • vaccine formulations utilize recombinant-made HIV particles as immunogens.
  • HIV vaccines have been prepared from attenuated or inactivated whole virions. Neither approach has been favored for the design of a vaccine against HIV-1 primarily because of the hazards associated with large scale preparation of the virus, potentially incomplete activation, and the introduction of the HIV genome into healthy recipients (Minor, 1989, J. Antimicrobial Chemotherapy 23, Supp. A: 55) . HIV vaccine development has therefore focused on subunit vaccine candidates. Although initial attention was directed to a subunit formulation comprising recombinant gpl20 envelope protein, it is now clear that both gpl20 and gp 41 are target antigens for the development of neutralizing antibodies (Chahn et al., 1986, EMBO J.
  • gp41 With cell membranes, in conjunction with its weak noncovalent interaction with gpl20, render impractical the purification of intact soluble gpl20/gp41 complexes. More importantly, like other viral antigens presented as components of membrane structures, such as Hepatitis B surface antigen (Cabral et al., 1978, J. Gen. Virol. 38: 339) and Herpes Simplex Virus glycoprotein (Ho et al., 1989, J. Virol. 63: 2951), membrane bound gpl20/gp41 complexes are likely to be more immunogenic than the soluble counterparts.
  • the present invention may circumvent many, if not most of the problems.
  • the recombinant-made HIV particles of the invention may be designed so that desirable epitopes are retained, while undesirable epitopes are deleted or modified.
  • the invention provides a means by which several different variable epitopes of the same HIV protein may be incorporated into the particles used in the vaccine formulation.
  • the invention also provides a way to create heterologous recombinant-made HIV particles that may be used to formulate a vaccine capable of preventing infection by both HIV-1 and HIV-2.
  • One of the novel aspects of a vaccination approach utilizing the present invention is that a full battery of these and other such epitopes may be consolidated into one immunogenic particle. Moreover, these epitopes are presented to the immune system as they are on native HIV, thereby inducing immune responses that are effective against infection by native virions.
  • HIV-1 VACCINES Protective immunity against HIV has not been fully o elucidated. It is commonly believed that both neutralizing antibodies and cell-mediated immunity may be required, since HIV can be transmitted in cell-free or cell-associated form.
  • Neutralizing antibodies recognizing a number of different HIV epitopes have been identified in HIV-1 infected 5 individuals, including those which recognize epitopes on highly conserved as well as variable regions of the envelope protein gpl20.
  • neutralizing antibodies directed against epitopes of gp41 and pl7 (Papsidero et al., 1989, J. Virol. 63:267-272) have been identified.
  • antibodies 0 detected in HIV infected individuals include those specific for domains of gpl20 which bind to the CD4 cell surface receptor. Still other antibodies, which bind to a hypervariable region of gpl20, are capable of inhibiting fusion of HIV infected cells into syncytia (Rusche et al., 5 1988, Proc. Natl. Acad. Sci. U.S.A. 85: 3198).
  • One embodiment of the invention is a vaccine against the HIV-1 virus, presently the most prevalent form of HIV, using recombinant-made HIV-1 particles such as the recombinant- made HIV-1 particles described in Section 6., et seq., infra, which comprise mature core and envelope proteins of the type 1 virus, assembled within a structure that mimics the morphologic and antigenic properties of live HIV.
  • This embodiment encompasses vaccine formulations using a variety of recombinant-made HIV-1 particles. For example, particles silmultaneously presenting the gpl20/gp41 complexes of two or more HIV isolates may be useful for inducing the development of protective antibodies against a number of variable region epitopes.
  • particles are designed to incorporate epitopes from HIV-1 strains having different tropisms.
  • recombinant-made HIV-l particles displaying determinants unique to a monocyte-associated HIV-1 strain in combination with determinants common to T4 cell-associated virus strains may be generated using a multi-infection approach.
  • such particles may be generated by coinfecting host cells with three recombinant vaccinia, one carrying the gag gene from one strain, one carrying the env gene from the same strain, and the other carrying the env gene from a tropogenically different strain.
  • the most effective vaccine against HIV-1 may involve using the recombinant-made HIV-1 particles of the invention in combination with other immunogens.
  • recombinant-made HIV-1 particles appear most effective at eliciting humoral and cellular immune responses when used as a secondary immmunogen following initial immunization with recombinant gpl60 in non-human primate subjects.
  • HIV-2 VACCINES AND HETEROLOGOUS VACCINES Other embodiments of the present invention relate to vaccines against HIV-2 as well as heterologous vaccines which may include, for example, a single vaccine for both HIV-1 and HIV-2.
  • the recombinant-made particles used in such heterologous vaccines may comprise, for example, the core proteins of HIV-1 and the envelope proteins of HIV-1 and HIV-2.
  • Vaccines comprising a single recombinant-made HIV particle type or different types in combination may be formulated with a suitable adjuvant in order to enhance the immunological response to their antigens.
  • suitable adjuvants include, but are not limited to, mineral gels, surface active substances such as lysolecithin, plurionic polyols, polyanions, peptides, oil emulsions, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
  • a number of methods well known in the art may be used to introduce the vaccine formulations described above, including intradermal scarification, intravenous injection, subcutaneous injection, intramuscular injection, intranasal administration, oral administration, etc.
  • Described here is an in vitro system for generating recombinant-made HIV-1 particles which contain assembled core and envelope proteins and which display the env gpl20 and gp41 antigens on their surfaces.
  • BSC-40 cells are coinfected with recombinant vaccinia viruses carrying either the complete envelope gene of HIV-1 (v-env5) or the complete HIV-1 gag and protease genes (v-gag2) . HIV-1 proteins are expressed in the BSC-40 cells and assemble into
  • HIV-1 particles which bud from the cell membrane.
  • the resulting particles are nonreplicating, react with monoclonal antibodies specific for HIV-1 envelope and core proteins, and are morphologically similar to HIV-1 virions.
  • African green monkey kidney cells (strain BSC-40, a continuous line of African Green Monkey Cells derived from BSC-1 cells, ATCC No. CCL26) were propagated in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum and 100 units per ml each of penicillin and streptomycin.
  • DMEM Dulbecco's modified Eagle's medium
  • Recombinant vaccinia viruses carrying HIV-1 env and gag gene sequences were prepared and evaluated as described in copending United States Patent Application Serial No. 905,217 filed September 9, 1986.
  • Recombinant vaccinia virus v-env5 carries the entire env gene of HIV-1.
  • Recombinant vaccinia virus v-gag2 carries the entire HIV-1 gag and prt genes as well as part of the pol gene.
  • the New York City Board of Health strain of vaccinia virus was purified from a commercial preparation of smallpox vaccine (Dryvax Lot 321501G) marketed by Wyeth Laboratories (Marietta, PA) .
  • Smallpox vaccine was diluted with PBSAM (see below) and plaque-purified three times successively on BSC-40 cells.
  • a stock hereafter designated as v-NY was prepared on BSC-40 cells from such a plaque-purified isolate and was used to construct recombinant viruses.
  • RIP radioimmunoprecipitation
  • EIA capture enzyme immunoassay
  • western blot analysis Western blot analysis
  • immuno electron microscopy Four techniques were used for the detection of HIV-1 env and gag proteins: radioimmunoprecipitation (RIP) , capture enzyme immunoassay (EIA) , western blot analysis, and immuno electron microscopy.
  • Radioimmunoprecipitations were performed essentially as described (Haffar et al., 1988, J. Cell. Biol. 107:1677) using human polyclonal anti-HIV-1 sera (Trimar, Inc.). Briefly, BSC-40 cells were infected with recombinant or parental vaccinia virus at a multiplicity of infection (MOI) of 10 PFU per cell per virus used. At 12 hours post- infection, the cells were radiolabeled for 4 hours with
  • Post-nuclear cell lysates or culture media were then reacted with the polyclonal antisera for 30 minutes at room temperature.
  • Antibody-antigen complexes were incubated with
  • Staph A 10% glutaraldehyde-fixed Staphylococcus aureus (Staph A) for 30 minutes at room temperature.
  • Staph A-antibody-antigen complexes were sedimented by centrifugation and washed twice with RIP wash buffer (1% NP-40, 0.1% SDS in PBS).
  • the resulting pellets were solubilized with SDS-PAGE sample buffer (Laemmli, 1970, Nature 227: 680), fractionated in 11.5% polyacrylamide gels, and the immunoprecipitated proteins visualized by autoradiography.
  • Capture enzyme immunoassay and western blots were used to specifically detect HIV-1 p24 as described (Hu et al. , 1987, Nature 328: 721) with a monoclonal antibodies against p24.
  • the monoclonal antibodies used for both capture enzyme immunoassays and Western blots were generated to p24 sequences by immunization of mice with recombinant fusion peptides that define overlapping sequences in the p24 protein.
  • Monoclonal antibodies 25-2 and 25-3 were generated at Genetic Systems (Seattle, WA) and are described in United States Patent Application Serial Nos. 054,026, filed April 30, 1987, and 105,761, filed October 7, 1987.
  • Immuno electron microcopy was used to detect gpl20 and gp41 envelope proteins on the surface of recombinant-made HIV-1 particles using MAb 110-4 specific for gpl20 (Thomas et al., 1988, AIDS Res. Hum. Retroviruses 2: 25; Linsley et al., 1988, J. Virol. 62: 3695) and MAb 41-1 specific for gp41 (Gosting et al., 1987, J. Clin. Microbiol. 25: 845), as well as to elucidate the morphology of mature recombinant- made HIV particles, following the electron microscopy techniques described in Section 6.1.3., infra.
  • ELECTRON MICROSCOPY Recombinant-made HIV-1 particles were isolated and prepared for EM visualization as follows: Culture media from 15 hr infected cell cultures were collected and pooled. The media was clarified from contaminating cells by centrifugation in a refrigerated table top centrifuge at 600 xg. The particulate fraction was then isolated from the clarified supernatants by ultracentrifugation at 120,000 xg. The resulting supernatant was collected as the post- particulate material (S) .
  • the particulate pellet was resuspended in phosphate buffered saline (PBS) pH 7.4 and resedimented by ultracentrifugation at 120,000 xg through a 15% sucrose solution layer.
  • PBS phosphate buffered saline
  • the twice sedimented material was refered to as the particulate fraction (P) .
  • the sedimented particulate pellet (P) was washed several times by gently overlaying the pellet with PBS and then aspirating it off. The pellet was then fixed with 4% paraformaldehyde for 20 minutes and washed again with PBS.
  • the fixed cells and fixed particulate pellets were then washed 5 times with PBS and then blocked with a solution containing 0.8% bovine serum albumin (BSA), 0.1% gelatin and 5% normal goat serum in PBS (blocking buffer) for 30 minutes. Blocking solution was decanted and monoclonal antibodies added to the cells as ascites fluid (diluted 1:2000 in blocking buffer) and allowed to incubate for 2 to 3 hours.
  • BSA bovine serum albumin
  • blocking buffer normal goat serum
  • Samples were then rinsed well with PBS, and dehydrated by 3 minutes sequential incubations in 35%, 50%, and 75% ethanol, prior to staining with 3% uranyl acetate in 70% ethanol for 30 minutes at 22*C. Samples were then further dehydrated with sequential incubations, as described above, in 80%, 90%, 95%, and finally 100% ethanol (3 times), at 22 ⁇ C.
  • the p55 gag precursor synthesized in v-gag2 infected cells is also processed to yield the mature gag proteins p24, pl7, pl5, as well as two intermediate precursor species p45 and p39 (lane E) (Gowda et al., 1989, citation). Interestingly, the gag proteins were also detected in the culture supernatant (lane F) .
  • BSC-40 cells coinfected with v-env5 and v-gag2 yielded processed env and gag proteins identical to those expressed in cells infected individually (lane C, compare with lanes A and E) .
  • the mature env and gag proteins generated by these doubly infected cells were similarly localized to the culture supernatants (lane D, compare with lanes B and F) with identical kinetics.
  • lactoperoxidase catalyzed iodination of plasma membrane-associated proteins revealed that the transport of gpl60, gpl20 and gp41 to the cell surface is the same in v-env5 infected cells as it is in doubly infected cells (Fig. 2, lanes A and B, respectively) .
  • culture supernatants were separated by into particulate (P) and post-particulate (S) fractions (Section 6.1.3., supra) and the HIV protein content of each fraction analyzed by RIP in parallel with unfractionated culture supernatants (TS) .
  • P particulate
  • S post-particulate
  • TS unfractionated culture supernatants
  • gpl20 derived from cells coinfected with v-env5 and v-gag2 was detected in the P fraction as well as the S fraction (FIG. 3(1), lanes E and F respectively), though the particulate- associated gpl20 contributed little to the overall level of detectable extracellular gpl20.
  • gp41 was detected only in the P fraction of supernatants from doubly infected cells (FIG.3(1), lanes E vs D and F) , indicating that gp41 is associated only with budding particles.
  • the gpl60 env precursor was detected in the P fraction of supernatants from doubly infected cells, albeit at low levels (FIG. 3(1), lanes E vs D and F) .
  • HIV-l core proteins expressed by infected BSC40 cells were analyzed similarly. All detectable core proteins— p24, p55, p45, p39, and pl7 — were observed in the P fractions from v-gag2 infected (FIG. 3(1), compare lanes H and I) as well as doubly infected (FIG. 3(1), compare lanes E and F) cell culture supernatants. P fractions obtained from doubly infected cells were also subfractionated by sedimentation through a continuous sucrose density gradient of 15%-60% and the presence of p24 antigen evaluated by EIA as described in Section 6.1.2., supra. The results depicted in FIG.
  • Intact BSC-40 cells coinfected with the two recombinant vaccinia viruses were similarly analyzed using the anti- 0 gpl20 MAb. Numerous 100-120 nm particles positive for the gpl20 antigen were visualized. The majority of these cell associated particles assumed either of two distinct morphologies, one characterized by a diffuse vesicle form (FIG. 4, "e”, open arrow) and the other distinguished by an 5 eccentrically-localized, thickened double-membrane region (FIG. 4, "d”, double arrow). Applicants speculate that these different structures represent various forms of immature particles which may parallel those occurring during HIV-l virion morphogenesis.
  • the nucleic acid content of the recombinant-made HIV-1 particles was determined by dot blot hybridization with P Q RNA probes reactive with either the gag or env sequences.
  • the probes were synthesized by in vitro transcription of DNA templates, carrying the gag or env sequences, in the presence of radiolabeled nucleotides using the Promega Riboprobe in vitro transcription kit according to 5 manufacturers directions. Briefly, recombinant particles.
  • RNA preparation lysis buffer (2M guandin isothiocyanate, 125mM sodium citrate pH 7.0, 0.125% sarkocinate, 50% dimethyl sulfoxide) and blotted onto nitrocellulose filters, in parallel with various concentrations of similarly solubilized Psoralin- inactivated HIV (equivalent to 2600ng, 260ng, 26ng, and 2.6ng p24) .
  • Separate filters were prepared for reaction with the gag specific or env specific probes.
  • nitrocellulose filters were incubated for 2 hour at 42*C in hybridization buffer (3x SSC, 50% Formamide, 5x Denhardt's solution, and 150ug nonspecific RNA) , prior to addition of the respective probes.
  • hybridization buffer 3x SSC, 50% Formamide, 5x Denhardt's solution, and 150ug nonspecific RNA
  • the filters were incubated with the probes overnight at 42*C, then washed extensively with O.lx SSC/0.1% SDS solution, air dryed, and analyzed by autoradiography.
  • a single viral vector approach involving the construction of a recombinant vaccinia virus containing both the env and gag genes of HIV-1 may be used.
  • a recombinant vaccinia virus containing HIV-1 env and gag genes is used to infect BSC-40 cells.
  • the infected cells express envelope and core antigens of HIV-1, which assemble in the infected cells to generate the recombinant HIV-1 particles.
  • Recombinant HIV-1 particles purified from the infected cell media are immunogenic in vivo.
  • V-G2E5 A RECOMBINANT VACCINIA VIRUS CONTAINING HIV-1 ENV AND GAG GENES
  • a 3.2 Kbp fragment which contains the entire env-coding sequence of HIV-1 (nucleotide no. 5707-8608) under the control of vaccinia virus 7.5K promoter was excised from plasmid pv-env5 (copending United States Patent Application Serial No. 07/593,401, filed October 5, 1990) by restriction endonuclease EcoRI.
  • Plasmid pv-gag2N contains the HIV-1 gag gene also under the control of vaccinia virus 7.5K promoter.
  • the construction of the resulting plasmid, pv- G2E5, is schematically represented in FIG. 6.
  • HIV-1 gag and env genes were introduced into the thymidine kinase gene of vaccinia virus by in vivo recombination as described in copending United States Patent Application Serial No. 07/593,401, filed October 5, 1990, thereby generating recombinant vaccinia virus V-G2E5, containing both gag and env genes of HIV-1.
  • African green monkey kidney cells (BSC-40) were infected with recombinant V-G2E5 at a multiplicity of infection of 5 pfu/cell. At 24 hr after infection, cells were washed and cell lysates were resolved by SDS-PAGE on a 7-15% gel.
  • V-G2E5 Recombinant V-G2E5 was used to produce HIV-l-like particles from BSC-40 cells. BSC-40 cells were seeded onto
  • Cytodex 3 beads (Pharmacia LKB Biotechnology) and were grown in spinner culture bottles in Dulbecco modified Eagle's medium with 5% fetal calf serum according to the manufacturer's recommendations. When cells attained confluency, they were infected with recombinant vaccinia virus V-G2E5 at a MOI of 5 pfu/cell. Following a 1 hr adsorption, the cells were washed twice with fresh medium to remove any excess inoculum. After 24 hr incubation at
  • FIG. 8 A radioimmunoprecipitation analysis of the particles produced by V-G2E5 infected cells is represented in FIG. 8. These results demonstrate the presence of both gpl20 and p24 antigens in the pellet fraction of infected cell medium after high-speed centrifugation, indicating the selective assembly of these mature virion proteins into particle forms. The relative abundance of p24 and gpl20 in these particles were similar to that of HIV-l virion preparations, underlying the structural and biochemical similarities between recombinant-made and authentic HIV-1 virions.
  • a system involving mammalian cells transfected with DNA encoding HIV-1 env and gag genes may be used. This system allows for the generation of recombinant HIV-1 particles in a stable mammalian cell line.
  • Generation of recombinant-made HIV particles using the system described herein may be accomplished using a variety of strategies, including but not limited to (1) the transfection of complex plasmid vectors containing multiple HIV structural or regulatory genes, (2) the co-transfection of multiple plasmid vectors containing different HIV genes, (3) the use of both constituitive and regulatable enhancer/promoter elements to drive the expression of HIV proteins, (4) the use of HIV regulatory proteins including tat and/or rev to indirectly control the expression of HIV gag and env structural proteins, (5) the use of different cell lines for expression of HIV proteins and recombinant- made HIV particles, and (6) the use of plasmid vectors encoding modified HIV proteins. These variations allow for the optimization of the system in different cell types and the manipulation of different protein species or structures in the recombinant HIV particles.
  • CmHi cytomegalovirus immediate early gene fused to the promoter and tar region of HIV (nucleotides -69 to + 78) .
  • Some of these plasmid vectors contain alternative enhancer:promoter elements derived from the cytomegalovirus immediate early gene (designated CMV) or the mouse metallothionein-I gene (Mt) .
  • the plasmid CmHiEnv ⁇ (1104-bl) has the Nrul to Hindlll fragement of pH3MPy, containing the Cytomegalovirus immediate early gene enhancer and HIV promoter and tar element, linked to nucleotides 5671-8572, Avail to Kpnl.
  • Plasmid CmHiTgfbEnv ⁇ (1113-al) contains the Nrul to Pstl enhancer:promoter fragment of pH3MPy linked to chimeric TGF-y3:HIV env gene; the simiam TGF-beta gene, providing the 5'untranslated region and signal peptide, is fused directly at the signal cleavage site of the HIV env gene, extending from nucleotide 5856 to the Kpnl site at nucleotide 8572.
  • Plasmid CmHiGag2Rre (1158-al) contains the Nrul to Xbal enhancer:promoter fragment of pH3MPy linked to HIV gag and pol coding sequences extending from the BssHII site at nucleotide 257 to the Asp718 site at nucleotide 3372. This is the same region of gag coding sequence contained in the recombinant vaccinia virus v-gag2, and includes the entire gag reading frame followed by about half of the pol reading frame. Also included in this region are sequences which encode the HIV protease and much but not all of the region encoding reverse transcriptase. An Xbal linker providing translational termination codons was ligated to the Asp718 site.
  • Plasmid CmvGag2Rre (1159-al) is identical except that the Nrul to Xbal cytomegalovirus enhancer and promoter fragment derived from the expression vector CDM8 (Invitrogen) was used.
  • Plasmid CmHiRev (1132-cl) contains the Nrul to Pstl enhancer:promoter fragment of pH3MPy upstream of an intronless HIV Rev gene, extending from the Bsu36I site at nucleotide 5500 to the Kpnl site at nucleotide 8572, with nucleotides 5590-7935 deleted.
  • Plasmid CmvRev (1152-al) is identical, except that the Nrul to Xbal cytomegalovirus enhancer and promoter fragment derived from the expression vector CDM8 (Invitrogen) was used.
  • Plasmid CmHiTat (1132-bl) contains the Nrul to Bglll enhancer:promoter fragment of pH3MPy upstream of an intronless HIV tat gene, extending from Sail site at nucleotide 5331 to the Kpnl site at nucleotide 8572, with nucleotides 5590-7935 deleted.
  • Plasmid BsMtRev (1202-2) contains a 1.7 kilobase fragment of the mouse metallothionein-I gene extending from an EcoRI site at approximately position -1700 to the transcriptional start site, upstream of an intronless HIV rev gene, extending from the Bsu36I site at nucleotide 5500 to the Kpnl site at nucleotide 8572, with nucleotides 5590- 7935 deleted.
  • Plasmid BsMtTat (1203-1) contains a 1.7 kilobase fragment of the mouse metallothionein-I gene extending from an EcoRI site at approximately position -1700 to the transcriptional start site, upstream of an intronless HIV tat gene, extending from the Sail site at nucleotide 5331 to the Kpnl site at nucleotide 8572.
  • Plasmid CmHIVdelXmn (1133-al) contains the same hybrid CMV:HIV Enhancer:Promoter derived from the expression vector pH3MPy used in plasmids described above.
  • This plasmid incorporates HIV 5' leader RNA sequences to the X nl site at nucleotide 384 just inside the N-terminus of the gag reading frame; contained within this segment is the splice donor site that is spliced to acceptor sites located upstream of many HIV genes, including those encoding the tat, rev and env proteins.
  • the Xmnl site at nucleotide 384 is joined through an Xbal linker (which contains a TAG translation' termination codon, stopping translation of a gag N-terminal peptide after about 20 amino acid residues) to an Xmnl at nucleotide 4034 near the C-terminus of the pol reading frame. HIV sequences then continue to the Kpnl site at nucleotide 8572, through the region encoding both HIV regulatory and minor structural proteins including vif, vpr, tat, rev, vpu, and for the HIV env protein.
  • This plasmid should be able to encode each of the HIV proteins listed above through splicing between the 5' splice donor site located just before the N-terminus of the gag gene and splice acceptor sites located upstream of the various protein coding reading frames.
  • the plasmid CmHIVdelKpnAvr(Gag2TRE) (1160-al) contains hybrid CMV-HIV Enhancer:Promoter driving expression of the entire gag reading frame and the N-terminal portion of the pol reading frame (contained in the recombinant vaccinia virus v-gag2) directly joined in the 3' portion of the HIV genome containing sequences encoding the tat, rev and env proteins.
  • Nrul to EcoRI Cytomegalovirus immediate early gene enhancer segment of expression vector P3MPy is linked to HIV promoter sequence begining at nucleotide -69; HIV leader RNA sequences continue through the gag gene and into the pol gene to the Kpnl site at nucleotide 3372, joined to a polylinker which includes an Nhel containing a TAG translation termination codon for the pol reading frame. This Nhel site is joined to an Avrll site at nucleotide 5207 in the vpr reading frame. HIV sequences then continue to the Kpnl site at nucleotide 8572.
  • This vector contains all of the sequence elements believed to important for the synthesis, splicing, cytoplasmic transport, translation, processing and function of HIV gag, protease, tat, rev, and env proteins.
  • This plasmid should be able to encode HIV gag from unspliced mRNA and for each of the other HIV proteins listed above through splicing between the 5' splice donor site located just before the N- terminus of the gag gene and splice acceptor sites located upstream of the various protein coding reading frames.
  • It also includes the tar element located within the first sixty nucleotides of the HIV primary mRNA transcript which is required for tat transactivation from the HIV promoter, and the Rre (rev responsive element) located between nucleotides 7315 and 7559 which is required for rev catalyzed cytoplasmic localization of mRNAs encoding HIV structural proteins.
  • this plasmid vector Not contained in this plasmid vector are a central region of the HIV genome encoding the C-terminal half of the pol reading frame, including part of the reverse transcriptase protein and all of the integrase protein, all of the vif reading frame and the N-terminus of the vpr reading frame; also absent from this vector is most of the 5' LTR and all of the 3' LTR as well as most of the nef reading frame.
  • Plasmid vectors CmHIVdelKpnAvr(Gag2TRE) and CmHiEnv ⁇ were transfected into dhfr- CHO cells. Transfected cell lines were selected by cotransfection with plasmid pSV2dl.fr.
  • CHO transfectants contained immunoreactive mature gag and env proteins, secreted in a particulate form with sedimentation properties similar to those of the HIV virion and the recombinant-made HIV-1 particles produced as described in Section 6., et seq., supra.
  • CHO cells (dhfr-) were transfected following growth in Ham's F12 nutrient mixture (without hypoxanthine) supplemented with 10% fetal bovine serum and 150 ⁇ g/ml proline in 60 mm tissue culture dishes to 50% confluency, transfer to serum free medium, incubation for 5 hours with a mixture of Lipofectin (BRL) , the HIV expression plasmids CmHIVdelKpnAvr(Gag2TRE) (1160-al) and CmHiEnvS (1104-bl) , and the selectable plasmid pSV2dhfr, removal of the Lipofectin:DNA mixture and transfer back to medium contianing serum. Two or three days post-transfection the cultures were transferred to selective medium, DMEM plus 10% FBS and proline. Two days later the cultures were trypsinized, and aliquots containing approximately 0.08% and
  • Tissue culture media from individual wells of the 96 well plates were then collected and assayed by a gag protein antigen EIA for secreted gag protein (Section 6.1.2, supra) .
  • a number of candidate wells were identified by this assay. Twelve potentially positive wells, four wells derived from each of three independent transfections, were then chosen for expansion. Visual insepection of positive wells at this point revealed that some wells were essentially confluent, others had only small colonies of growing cells, and others apparently contained no viable cells. Cells from all three classes of potentially positive wells were expanded; wells were trypsinized and cells transferred to 6 well dishes for expansion.
  • a small fraction of the cells from each candidate cell line was seeded into duplicate wells of a 24 well plate. After five days of growth, wild type HeLa cells or HeLa T4 cells (transfected with and expressing the CD4 protein) were added to each well. The following day, gag expression and env function was assayed by a focal immunoassay. Briefly, cells were fixed and incubated with a human anti-HIV serum, followed by a horseradish peroxidase linked goat anti-human antibody, and a peroxidase substrate (3-amino-9-ethyl-carbazole) . Four cell lines, falling into two classses, were positive by this assay.
  • cell lysates were analyzed by Western blot for HIV protein synthesis.
  • Cells in 60 mm or 100 mm tissue culture dishes were washed twice with PBS and collected directly into 300 ⁇ l or 750 ⁇ l of Laemmli sample buffer. Afer boiling, the total cellular protein in the sample was resolved by electrophoresis on a 10% polyyacrylamide gel or an 8-16% gradient polyacrylamide gel. Aliquots of HIV, CEM cells infected with HIV, and BSC-40 cells infected with vaccinia recombinants v-gagl, v-gag2, or v-env5 were included as controls.
  • the contents of the gel were electro-transferred to a sheet of nitrocellulose filter.
  • the filter was reacted with either AIDS patient serum or serum from a rabbit immunized with gpl60 derived from v-env5 infected cells, extensively washed, reacted with 125-1 labeled Protein A, and extensively washed again. The products were then visualized by autoradiography with X-ray film.
  • the four positive lines identified in the previous assay were also found to be positive for gag proteins.
  • 3010-C1 and 3010-C6 lysates contained several-fold more gag than did 3010-C5 and 3010-C13 lysates.
  • Lines that were negative in the previous assay were also negative for gag by Western blot.
  • Most of the immunoreactive material co- migrated with the p55 precursor and as a slighter smaller species not corresponding to a major product in HIV or in cells infected with HIV or v-gagl; only very minor amounts of mature p24 and pl7 gag proteins were detectable in cell lysates.
  • This analysis also demonstrated the presence of gpl60, gpl20 and gp41 envelope proteins in cell lysates of the positive cell lines.
  • gag and env proteins synthesized in these cells were assembled into virus-like particles and secreted
  • culture supernatants were collected and cleared by low speed centrifugation.
  • the particle fraction was collected by centrifugation (either 32,000 rpm in the SW55 rotor, 27000 rpm in the SW41 rotor, or 19000 rpm in the Type 19 rotor) .
  • Western blot analysis demonstrated that this material contains both gag and env proteins.
  • the primary gag proteins detected in the particles are the mature p24 and pl7 species; smaller amounts of the p55 and p40 precursors could also be detected. In contrast, precursor species accounted for most of the material in cell lysates.
  • Gpl60, gpl20, and gp41 envelope proteins were all detected in the particles. Comparison of Western blots suggests that only a small fraction of the gag and env proteins contained in the cells are secreted as particles.
  • Quantitative gag antigen EIA analysis of multiple 3010-C6 cell-derived particle preparations suggested yields of 3-17 ng gag per ml of culture medium. Particles from the 3010-C6 cell line were also analyzed by sedimentation into a sucrose gradient (2 hours at 50,000 rpm in the SW55 rotor, 15-60% sucrose). As shown in FIG. 10, both gag antigen EIA and Western blot assay of fractions from the gradient showed that the particles banded in a single peak at approximately 35-40% sucrose.
  • Plasmids and combinations of plasmids were tested by transient transfection into HeLa (and in a few cases BSC-40 and Vero) cells by standard calcium phosphate transfection procedures. Products were analyzed by polyacrylamide gel electrophoresis of total cell lysates or of recombinant-made HIV particles collected from culture medium by high speed centrifugation, electro-transfer to nitrocellulose filters, and probing with specific anti-sera.
  • the filter was probed with 125-1 labeled monoclonal antibody 110-4 (Section 6.1.2., supra) , which binds an epitope in the V3 region of gpl60 and gpl20; for analysis of gag proteins the filter was probed with AIDS patient serum (TriMar) followed by 125-1 labeled Protein A. The products were then visualized by autoradiography with X-ray film.
  • HIV gag and env proteins can be obtained by transfection of complex plasmids encoding multiple HIV proteins, including both structural and regulatory proteins; alternatively, multiple plasmids encoding different HIV proteins can be transfected together.
  • Transfection into HeLa cells of plasmids coding individually for env (CmHiTgfbEnv ⁇ or CmHiEnv ⁇ ) , or for gag (CmHiGag2Rre) , in combination with plasmids encoding tat and rev proteins results in the expression of immunoreactive gpl60 and pgl20 HIV envelope proteins or in the expression of immunoreactive HIV gag related proteins including the p55 primary translation product, p47 and p39 processing intermediates, and p24 and pl7 mature gag proteins.
  • transfection into HeLa cells of plasmid vectors (CmHIVdelXmn or CmHIVdelKpnAvr(Gag2TRE) ) , which contains in a single transcriptional unit the functional coding sequences for the both the tat and rev regulatory proteins and the env, or both env and gag, structural protein(s) also results in the synthesis of both precursor and mature env or gag and env proteins.
  • Co-transfection of CmHIVdelXmn with CmHiGag2Rre or CmVGag2Rre also results in the synthesis of both gag and env proteins.
  • Each of the plasmids or combinations CmHIVdelKpnAvr(Gag2TRE) (1160-al) , CmHIVdelXmn (1133-al) + CmHiGag2Rre (1158-al) , and CmHIVdelXmn (1133-al) + CmvGag2Rre (1159-al) also results in the secretion of recombinant-made HIV particles into the culture medium.
  • gag and env structural proteins and the production of recombinant-made HIV particles using this system may be achieved by multiple routes. Comparisons of the levels of expression achieved with different plasmid combinations are shown in Table II, below, demonstrating that different combinations may be optimal for the expression of different proteins. For example, env appears to be more efficiently expressed when co-transfected with plasmids coding separately for tat and rev proteins.
  • gag may be more efficiently expressed from a plasmid such as CmHIVdelKpnAvr(Gag2TRE) (1160-al) , which contains in a single transcriptional unit the functional coding sequences for both regulatory proteins. Intermediate levels of both proteins were obtained by co-transfection of the complex plasmid CmHIVdelKpnAvr(Gag2TRE) (1160-al) and a separate env coding plasmid.
  • gag and env expression levels were determined independently, and are not necessarily represented by the same scale.
  • rev is important for the efficient cytoplasmic localization of mRNAs encoding HIV structural proteins, and is dependent on a cis-acting regulatory element, termed the rev responsive element (Rre) , located within the portion of the HIV env gene encoding the N- terminal portion of the gp41 molecule, which was introduced into the gag plasmids as a Bglll to Hindlll fragment from the envelope gene, and may be intrisinically necessary for efficient expression of HIV gag and env proteins.
  • the tat protein is important for enhancing transcription initiated from the HIV promoter by its interaction with a cis-acting regulatory element, called tar, located within the first 75 nucleotides of HIV RNA transcripts.
  • the tar gene is present in the CmHi hybrid enhancer element utilized by CmHiGag2Rre, CmHiTgfbEnv5, and CmHiRev.
  • Expression of gag or env can be made independent of tat by co-transfection of plasmids such as CmvGag2Rre and CmvRev, since these plasmids do not contain the tar region.
  • gag expression from transfection of CmvGag2Rre + CmvRev is lower than from transfection of CmHiGag2Rre + CmHiTat - CmHiRev.
  • a cell line producing recombinant- made HIV particles may be further transfected with a plasmid encoding a minor HIV protein such as vpu or vif to alter the production levels and/or properties of the recombinant-made HIV particles.
  • cell lines transfected with a plasmid encoding tat, rev and gag proteins which therefore produce particles containing only gag proteins, could be selected and subsequently transfected with different env coding plasmids to generate particles containing env proteins from different strains or having other strutural alterations.
  • CMHIEnvS natural signal sequence of the HIV env genes
  • CmHiTgfbEnv ⁇ a fusion between the signal sequence of simian TGF-beta-1 and the N-terminus of mature gpl60
  • dhfr- CHO cells were transfected with the plasmid combination CmHiTgfbEnv ⁇ + CmHiTat + CmHiRev, along with the selectable plasmid pSV2dhfr.
  • Initial examination of cell lines selected in this experiment did not reveal detectable levels of HIV env expression.
  • Cells were then further selected by growth in increasing levels of methotrexate for amplification of the transfected selectable pSV2dhfr plasmid and co-amplification of the HIV tat, rev and env coding plasmids in the hope that this would select for amplification of low levels of HIV env expression.
  • One cell line was identified in which HIV env proteins were being synthesized.
  • plasmids utilizing regulatable promoters controlling the expression of HIV proteins.
  • Such plasmids can be transfected into cells which would initially be grown in the uninduced state, then be induced to high levels of HIV protein expression to allow short-term high levels of production before the onset of the toxic effect.
  • plasmids individually encoding tat and rev proteins under the control of the metal regulated mouse metallothionein-I promoter were constructed.
  • regulatable promoters can be used to modulate the expression of HIV proteins, and that the expression of HIV structural proteins can be indirectly regulated by modulation of the HIV tat and rev regulatory proteins.
  • Other examples of this approach include the use of regulatable promoters to control expression from complex plasmids encoding multiple HIV proteins, or the construction of novel inducible regulatory elements, for example, such as a hybrid Mt:Hi enhancer promoter containing the metal regulatory elements of the metallothionein promoter linked to the promoter and tar region of HIV.
  • CmHIVdelKpnAvr(Gag2TRE) were transfected into HeLa, BSC-40 and Vero cells.
  • the basic pattern of expression of gag and env, both with respect to the products made and expression efficiency are similar in all three cell lines. However, overall expression levels are highest in HeLa cells, approximately 3-fold lower in BSC-40 cells, and another 5- fold lower in Vero cells.
  • vaccinia viruses Two recombinant vaccinia viruses which direct the expression of truncated HIV-1 gpl60 in infected BSC-40 cells are described below. These recombinant vaccinia viruses may be used as vectors, in conjunction with v-gag2 (Section 6., et seq., supra) or other core antigen-encoding vectors, for generating recombinant HIV-l particles which may have enhanced anti-viral and/or immunogenic properties using the system described in Section 6., supra.
  • a recombinant plasmid was constructed which contained the HIV-1 env encoding sequence from nucleotide numbers 5705 to 8068 inserted into vaccinia recombination vector pGS62 (copending United States Patent Application Serial No. 07/593,401 filed October 5, 1990) at the BamHI site downstream from the 7.5K promoter.
  • the HIV-1 sequence was derived as a 2.36 Kbp BamHI fragment from plasmid pv-env5 (copending United States Patent Application Serial No. 07/593,401, filed October 5, 1990).
  • the fragment contained the entire coding sequence of gpl20 and the N-terminal 241 amino acids of gp41, including 49 amino acid residues of the cytoplasmic region of gp41 at the C-terminus of the proposed transmembrane sequence.
  • the chimeric gene containing the 7.5K promoter and the HIV-1 env sequences was inserted into the vaccinia virus thymidine kinase gene according to procedures described in copending United States Patent Application Serial No. 07/593,401, filed October 5, 1990.
  • the resultant recombinant virus V-ED2 directs the expression of a truncated gpl60 which is cleaved into gpl20 and gp41 as efficiently as wild-type gpl60 (FIG. 12) .
  • HeLa CD4 cells infected with V-ED2 formed syncytia (multinucleated giant cells, characteristic of HIV-1 infection) more readily than v-env5, which expresses a full-length gpl60 envelope glycoprotein precursor.
  • V-ENV5DCT Recombinant virus V-ENV5DCT was constructed to contain the entire env coding sequence of HIV-lv (BRU isolate; Wain-Hobson et al., 1985, Cell 40:9317), except for the C- terminal 13 amino acids of gp41.
  • the deletion mutation was introduced by an oligonucleotide mutagenesis procedure as described (Kunkel et al., 1987, Meth. In Enzymol. 154:367- 382).
  • the chimeric gene containing the 7.5K promoter and the mutated HIV-1 env sequences was inserted into the vaccinia virus thymidine kinase gene according to procedures described in copending United States Patent Application Serial No. 07/593,401, filed October 5,1990.
  • EXAMPLE GENERATION OF RECOMBINANT HIV-1 PARTICLES HAVING MODIFIED STRUCTURAL CHARACTERISTICS USING RECOMBINANT VACCINIA VIRUSES EXPRESSING UNPROCESSED GP160
  • Two recombinant vaccinia viruses which direct the expression of a gpl60 precursor envelope glycoprotein having mutations in the proteolytic cleavage site(s) between gpl20 and gp41 are described herein.
  • recombinant vaccinia viruses may be used as vectors in conjunction with v-gag2 or other core-antigen-encoding vectors for generating recombinant HIV-1 particles which may have enhanced antiviral and/or immunogenic properties using the system described in Section 6., supra.
  • pv-160NC A recombinant plasmid (pv-160NC) was constructed that contained a mutated gpl60-coding sequence from nucleotide numbers 5803 to 8495 inserted downstream from the 7.5K promoter in vaccinia recombination vector pGS62.
  • the mutations were introduced by oligonucleotide-directed mutagenesis essentially as described (Kunkel et al., 1987 (Meth. Enzymol. 154:367-382). The mutated sequences are shown below ("t" represents cleavage site between gpl20 and gp41) :
  • the chimeric gene was inserted into the vaccinia virus genome at the thymidine kinase gene by in vivo recombination as described in copending United States Patent Application Serial No. 07/593,401, Filed October 5, 1990.
  • the resulting recombinant virus directs the expression of a gpl60 precursor envelope glycoprotein that is not cleaved into gpl20 and gp41.
  • HIV-1 env gene as V-160NC (Section 10.1., supra) , but under the control of vaccinia virus UK promoter was constructed.
  • a 2.26 Kbp BamHI fragment was excised from plasmid pv-160NC and was inserted into a derivative of vaccinia recombination vector pSCIO (Chakrabarti et al., 1985, Mol. Cell. Biol. 5:3403-3409) at the EcoRI site downstream from the UK promoter. This generated a chimeric gene containing the vaccinia virus UK promoter and the entire coding sequence for the mutated gpl60. The chimeric gene was inserted into the vaccinia virus genome at the thymidine kinase gene. The resultant recombinant virus directs high level production of mutant HIV-1 gpl60 in infected cells.
  • vpu This protein is a non- glycosylated polypeptide of 16 Kd apparently associated with the inner surface of the cytoplasmic membrane.
  • vpu is not required for particle formation, mutations in vpu result in a decrease in virions released from infected cells (Terwilliger et al, 1989, Proc. Natl. Acad. Sci. U.S.A. 86:5163-5167; Strebel et al., 1989, J.
  • vpu may play a role in facilitating the process of virion assembly and/or release.
  • the following example describes a system with which the potential role of such accessory molecules could be examined and their utility in recombinant particle production demonstrated.
  • a recombinant vaccinia virus was constructed that contained the entire HIV-1 vpu-coding sequence.
  • a 290-bp fragment of HIV-1 cDNA from nucleotide numbers 5636-5927 was inserted into vaccinia recombination vector pGW62 between the Smal and EcoRI sites downstream from the 7.5K promoter. This fragment contains the coding sequence of vpu as well as 7 bp of 5'- and 37 bp of 3'-untranslated sequences.
  • the chimeric gene was then inserted into the vaccinia virus genome by in vivo recombination.
  • the effect of vpu on recombinant particle formation and release can be demonstrated in cells co-infected with v-vpu and V-G2E5 as described in Section 7, et seq., supra.
  • This example demonstrates the ability of the recombinant-made HIV-1 particles of the invention to reduce or abrogate the infectivity of CD4 + lymphocytes by HIV m vitro.
  • the results indicate that recombinant-made HIV-1 particles effectively inhibit HIV-1 infection in a dose-dependent manner.
  • CEM T-lymphoblastoid cells
  • Control wells received no particles but were corrected for volume by the addition of 0.4 ml media. Cells and particles were allowed to incubate at 37 " C for 3 hours prior to addition of HIV. A set of duplicate wells corresponding to each of the various particle concentrations received (1) no
  • HIV input (2) low virus input of 40 TCID-.- (tissue culture infectious dose units 50) corresponding to 5pg p24 gag, or (3) high virus input of 400 TCID_ , corresponding to 50pg p24.
  • the culture media was withdrawn from the wells and replaced with fresh media containing the appropriate original concentration of particles.
  • aliquots of cells were collected from all wells and assayed for infectivity by determining intracellular expression of HIV antigens using an indirect immunofluorescence assay employing human polyclonal anti-HIV sera as primary antisera, followed by goat anti-human IgG fraction conjugated to fluorescene as secondary sera.
  • VIRUS FLUORESCENCE % ( TCID 5 ⁇ I DAY 5 DAY 6
  • HIV-1 Human Immunodeficiency Virus Type 1 (HIV-1) , strain LAV-1
  • PBLs used in this system are isolated from HIV-1 infected seropositive donors, and the frequency of infected cells within the isolated PBL fraction ranges from 0.04% - 1.3%
  • Peripheral blood lymphocytes were harvested from heparinized blood samples of HIV-1 seropositive donors by fractionation of the buffy coat material over a Hypaque- ficoll cushion. The cells were then incubated in culture media (RPMI-1640 supplemented with 10% Human Serum) , and the CD8 lymphocytes were depleted by treatment with CD8, CD16, and CD20 specific monoclonal antibodies for 1 hr at 37"C followed by the addition of rabbit complement for 1 hr at 4°C (Zarling, J.M. et al., 1990, Nature (London) 347: 92- 95) . The resulting cell preparation consisted mainly of CD4 lymphocytes, B lymphocytes and macrophages.
  • the cells were seeded into 24 well plates at IM cells/well in 0.5 ml culture media. Duplicate wells received varying concentrations (see Tables IV and V) of either recombinant- made HIV-l particles, or psoralen/U.V.-inactivated HIV-l virions. All wells were then supplemented with 0.5 ml of culture media containing MAb G19.4 reactive with T cell CD3 antigen (Hoxie, J.A., et al. 1986 Science 234:1123-1127) at lug/ml final concentration as well as interleuken 2 (IL-2) at a final concentration of 5%.
  • MAb G19.4 reactive with T cell CD3 antigen Hoxie, J.A., et al. 1986 Science 234:1123-1127
  • IL-2 interleuken 2
  • Activation of T cells with a soluble CD3 MAb has been shown to induce the replication of latent HIV-1 virus (Zarling, J.M. et al., 1990, Nature (London) 347: 92-95).
  • donors Z29,Z30 or donors Z31,Z39 respectively the culture media was withdrawn from the wells and the cells were washed with PBS supplemented with 10% human serum and replaced with fresh culture media.
  • Identical samples were either allowed to incubate with the fresh media alone (see below) or were again supplemented with the appropriate concentrations of recombinant-made HIV-1 particles or inactivated HIV-1 virus.
  • samples of culture media were harvested from the wells and analyzed for p24 gag content by specific EIA (Section 6.1.2., supra) . Additionally, cells were concomitantly collected and examined for HIV-1 protein expression by indirect fluorescence using a mixture of monoclononal antibodies reacting with gpl20 and p24 gag . Cells that had the recombinant-made particles or inactivated virus washed out on day four or five, were passaged on day 8 into new wells containing immobilized anti-CD3 MAb to induce continued replication of the cells.
  • the results of the infectivity assays are presented in Tables IV and V.
  • the recombinant-made HIV-1 particles inhibited the spread of virus infection through the culture in samples derived from four different seropositve patients. This inhibition was dose dependent and was detectable at 0.1 / .g/ml eqivalent of p24 TM " protein in samples treated with recombinant-made HIV-1 particles.
  • the psoralen/U.V. inactivated virus similarly inhibited the spread of virus infection in the cultures, in some instances (Table IV, donor Z29, day 6 samples), 10-fold more inactivated virus was needed compared to the recombinant- made HIV-1 particles.
  • the humoral immune response of each immunized rabbit was evaluated by ELISA and Western Blot analysis.
  • the ELISA allowed the measurement of the overall HIV specific antibody titer, while the western blot analysis elucidated antibody reactivity with individual viral proteins.
  • the cellular immune response elicited in two of the immunized rabbits was characterized. Results of these experiments confirmed the ability of the recombinant made HIV-1 particles to generate HIV-specific immune responses in immunized animals.
  • the immunogens were formulated with Complete Freunds adjuvent at 1:1 ratio and administered by intramuscular injections at amounts equivalent to 120ug p24 gag and 4-6ug gpl20 env proteins.
  • the rabbits received secondary immunizations (2°, boost) by the subcutaneous route of material formulated in Incomplete Freunds adjuvent also at a 1:1 ratio.
  • the amount of immunogen used in the 2° immunization was equivalent to 190ug p24 and 10-20ug gpl20 proteins (rabbit 238 and 239) , or 100 ⁇ g p24 and 5 to 10 ⁇ g gpl20 protein (rabbits 241 and 243) (see Table VI) .
  • the filter was subsequently divided into identical strips and used in the Western blot analysis. Briefly, following blocking of the strips for 3 hours with blocking buffer (3% dried milk in PBS, BLOTTO) at 22"C, each strip was incubated with a 1:50 dilution, in BLOTTO 0.2% NP40, of specific rabbit serum sample overnight at 4"C. The strips were then washed extensively, and incubated with 125I Protein A (ICN).
  • blocking buffer 3% dried milk in PBS, BLOTTO 0.2% NP40
  • Protein bands were visualized by autoradiography. The sera were also tested for the presence of neutralizing antibodies that reduce the level of p24 a production, an indicator of virus production and release.
  • Presence of antibodies that neutralize the LAV-BRU isolate in the sera of the immunized rabbits were detected by using a ⁇ n vitro CEM cell infectivity assay.
  • Sera were heat inactivated and then serially diluted in culture medium (RPMI-1640 supplemented with 10% Fetal calf serum) .
  • Equal volumes of diluted serum and 30 TCID_. n of virus inoculum were mixed and incubated at 37° for 45 min.
  • the challenged virus preparation (0.1ml) was added to 0.1ml cultures of CEM cells (2x10 4 cells) m 96-well mi.crotiter plates and incubated at 37°C with occasional mixing.
  • the virus-serum mixture was removed from the wells and replaced with culture medium containing the original rabbit serum at the appropriate diultion. After 5 days, the progress of the infection in the cultures was monitored by measuring the p24 gag levels in the supernatants by EIA.
  • FIG. 14 presents the relative antibody titers in serum samples collected prior to (Prebleed) , as well as 2, 5, and 7 weeks following the 1° immunization as determined by ELISA for rabbits 238 and 239.
  • Rabbit 238 (FIG. 14, graph A) exhibited a hightened immune response to HIV proteins after 5 weeks which was maintained following the 2" immunization (see week 7 data, i.e. 2 weeks post-boost) .
  • week 7 data i.e. 2 weeks post-boost
  • substantial reactivity with HIV proteins was detected 2 weeks folowing 1° immunization in rabbit 239 samples (FIG. 14, graph B) , and diminished by 5 weeks.
  • a prominent boosting effect in antibody titer was observed in serum sample collected from rabbit 239 at week 7.
  • FIG. 15 are the results of the ELISA assays conducted on all of the rabbit sera using either immobilized whole disrupted virus (panels 1 and 2) or immobilized purified envelope glycoprotein gpl20 (panels 3 and 4) .
  • the data presented are the end point titers at different intervals following the primary immunization.
  • the abscissa values represent the weeks post-primary immunizations when serum samples were collected.
  • the arrows indicate the times of the secondary and tertiary immunizations (see Table VI) .
  • both rabbits immunized with the recombinant-made HIV-1 particles (R238 and R241) as well as the rabbits immunized with the psoralen/U.V.
  • inactiviated HIV-1 virions (R239 and R243) generated a profile of antigen reactivity that correlated with the boost schedule.
  • the titer of antibodies in the various rabbit sera reactive with the disrupted whole virus was primarily a reflection of the reactivity with the p24 ga protein, which constitutes approximately 90% of the protein content in both recombinant-made HIV-1 and HIV-1 virions.
  • Reactivity to the whole virus was approxmiately two to three orders of magnitude higher than reactivity to gpl20, a disparity directly related to the low levels of the envelope glycoproteins (5-10%) in the particle structures.
  • FIG. 16 shows that immunization with the recombinant- made HIV-l particles generated high titers of neutralizing antibodies to homologous HIV-1 (LAV-1 strain, BRU isolate) (panel A) .
  • the titer of the neutralizing antibodies correlated with the boost schedule.
  • the titers reported correlate with 75% inhibition in p24 ga production.
  • Stimulation index cpm [H]TdR incoroporated into stimulated PB1, divided by cpm [H]TdR incorporated into stimulated PBL
  • FIG. 17 presents the results of an analysis of the reactivity of immunized rabbit sera on viral proteins by Western blot.
  • the results of the Western blot analyses are presented in FIG. 8.
  • the primary immune response in week 5 serum from rabbit 238 was directed against the gag proteins, with only gp41 env protein reactivity (FIG. 17, lane 1) .
  • FIG. 17, lane 1 The results of the Western blot analyses show that there was redistribution of antibody response between the various gag proteins, and the emergence of new antibody reactivity with the higher molecular weight HIV proteins (Fig. 17, lane 2).
  • Lanes 5, 6 and 7 of FIG. 17 represent reactivity of various concentrations (1:100, 1:1000, and 1:10000, respectively) of pooled human sera from seropositive individuals and serve as control antibody.
  • Described herein is an experiment designed to test whether the recombinant-made HIV-1 particles of the invention bind to CD4 cells and enter by fusion wit plasma membrane as do HIV-1 virions.
  • HeLa cells transfected with the gene encoding the CD4 molecule are used in this assay. These cells have been extensively characterized and have been shown to support productive HIV-1 infection. Cells were grown in monolayers on a glass slide in Dulbecco's modified Eagles medium supplemented with 10% fetal calf serum. The cells were washed with PBS/1%FCS and incubated with 50 ⁇ g/ml recombinant-made HIV-1 particles for 2 hr at 37"C. The cells were then washed extensively and fixed with 3.7% paraformaldehyde solution.
  • FIG. 18A shows a HeLaCD4 cell exhibiting positive fluorescence following incubation with the recombinant-made HIV-1 particles.
  • FIG. 18B shows the same cell optically sectioned by CLSM, starting from the top of the sample and progressing towards the slide surface in l,.m increments. The micrograph panels show that the fluorescence increases as more of the intracellular environment is revealed, indicating that the recombinant- made HIV-1 particles were internalized following binding.
  • the following examines the immunogenicity of recombinant-made HIV-1 particles in a non-human primate species immunized with recombinant-made HIV-1 particles, psoralen/UV-inactivated HIV-1 virions, and recombinant vaccinia virus expressing HIV-1 gag and env antigens.
  • the results demonstrate that the recombinant-made HIV-1 particles elicited both cell-mediated and humoral immune responses, including neutralizing antibodies to HIV-1.
  • IMMUNIZATION PROTOCOL Twelve macaques were immunized with recombinant-made HIV-1 particles, psoralen/UV- inactivated HIV-1 virions, or recombinant vaccinia virus expressing HIV-1 gag and env antigens, according to the following schedule:
  • Each dose of recombinant-made HIV-like particles and psoralen/UV-inactivated HIV virions contained an equivalent of 200 ⁇ g of p24 as determined by p24 antigen capture assay (Genetic Systems) and approximately 6 ⁇ g of gpl20 as determined by immunoblot assay.
  • Recombinant gpl60 was purified from BSC-40 cells infected with recombinant vaccinia virus expressing the same antigen and was used at 6 ⁇ g per animal per immunization, a dose level similar to that presented by the recombinant-made HIV-1 particles.
  • All particle or subunit immunogens were formulated either in Freund's incomplete adjuvant (Difco) or in DETOX (RIBI Immunobiochem) , which containted detoxified monophosphoryl lipid A and bacterial cell wall skeleton. All immunization with recombinant-made particles, inactivated HIV-1 virions, or recombinant gpl60 were performed by intramuscular injections.
  • Antibody response was determined at 4 weeks after the secondary immunization, i.e. week 12 for animals in Groups 1 and 2 and week 8 for animals in Groups 3 to 6.
  • Lymphoproliferative response was determined at 4 weeks after the primary immunization.
  • Stimulation index (S.I.) was determined by dividing the c.p.m. H-thymidine incorporated into HIV-1-stimulated cells by the c.p.m. incorporated into non-stimulated cells.
  • recombinant-made HIV-1 particles are immunogenic and can elicit an HIV-specific immune response, particularly when used in animals previously primed with recombinant vaccinia virus expressing both env and gag antigens; (2) boosting previously primed animals with recombinant-made HIV-1 particles was more effective at eliciting an immune response than boosting with equivalent amounts of soluble gpl60, most likly due to the effective presentation of envelope antigens by the recombinant-made HIV-1 particles; and (3) when used as the sole immunogen for primary and secondary immunizations, recombinant-made HIV-1 particles elicit HIV- specific antibody and T-cell immune responses in the majority of immunized macaques at levels similar to those immunized with equivalent does of inactivated HIV-1 virions.
  • nucleotide positions given for HIV-1 genes are based on the genomic sequence of the LAV-BRU isolate, Genebank accession no. K02013 (Wain-Hobson et al., 1985, Cell 4_0:9317) .

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Virology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Sont décrites des particules rétrovirales non réplicantes produites par recombinaison possédant des caractéristiques structurales, morphologiques et immunologiques très semblables à celles des rétrovirus d'origine humaine. La méthode de l'invention implique la coexpression du noyau rétroviral arrivé à maturité et des protéines structurales de l'enveloppe dans les cellules hôtes chez les mammifères, de sorte que les protéines rétrovirales exprimées s'associent en un amas de particules rétrovirales naissantes. Dans une forme d'excécution particulière de cette invention, les particules non réplicantes de VIH-1 produites par recombination sont obtenues en coinfectant des cellules hôtes de mammifères avec un virus vaccinal de recombinasion porteur de gènes gaz de protéase du virus d'immunodéficience humaine du type 1 (VIH-1) et d'un virus vaccinal de recombinaison porteur du gène env du VIH-1. Ces particules de VIH-1 non replicables produites par recombinaison ont des caractéristiques immunologiques et morphologiques se rapprochant de très près de celles du VIH-1 d'origine, elles sont capables de bloquer le caractère infectieux du VIH vivant in vitro, et elles sont fortement immunogéniques in vivo. Les particules de VIH-1 produites par recombinaison décrites, peuvent être employées comme agents antiviraux et comme immunogènes dans des formulations de vaccins efficaces pour inhiber ou pour empêcher l'infection par le VIH et/ou le développement du syndrome d'immunodéficiance acquise.
EP19910900526 1989-11-20 1990-11-20 Non-replicating recombinant-made retroviral particles used as antiviral agents and immunogens Withdrawn EP0502105A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US43920589A 1989-11-20 1989-11-20
US439205 1999-11-12

Publications (2)

Publication Number Publication Date
EP0502105A1 true EP0502105A1 (fr) 1992-09-09
EP0502105A4 EP0502105A4 (en) 1993-02-24

Family

ID=23743729

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19910900526 Withdrawn EP0502105A4 (en) 1989-11-20 1990-11-20 Non-replicating recombinant-made retroviral particles used as antiviral agents and immunogens

Country Status (14)

Country Link
EP (1) EP0502105A4 (fr)
JP (1) JPH05503629A (fr)
KR (1) KR920703639A (fr)
AU (1) AU636944B2 (fr)
CA (1) CA2068713A1 (fr)
ES (1) ES2052478T1 (fr)
FI (1) FI922277A (fr)
GR (1) GR930300005T1 (fr)
HU (1) HUT60506A (fr)
NO (1) NO921969L (fr)
OA (1) OA09698A (fr)
TW (1) TW216446B (fr)
WO (1) WO1991007425A1 (fr)
ZA (1) ZA909302B (fr)

Families Citing this family (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU5848390A (en) * 1989-06-01 1991-01-07 Applied Biotechnology, Inc. Self-assembled, defective, non-self-propagating viral particles
US5861282A (en) * 1989-10-16 1999-01-19 Whitehead Institute For Biomedical Research Non-infectious HIV particles and uses therefor
WO1991019803A1 (fr) * 1990-06-19 1991-12-26 Applied Biotechnology, Incorporated Particules virales autoassemblees defectueuses qui ne se propagent pas d'elles-memes
DE4124817A1 (de) * 1991-07-26 1993-01-28 Basf Ag Waessrige polymerisatdispersionen
WO1993009238A1 (fr) * 1991-11-08 1993-05-13 The Upjohn Company Vaccins contre le virus de la leucemie feline
JPH07505412A (ja) * 1992-04-14 1995-06-15 ブリティッシュ　バイオテック　ファーマシューティカルズリミテッド Ｃｔｌ応答の誘導
ATE420193T1 (de) 1993-01-26 2009-01-15 Univ Pennsylvania Zusammensetzung zur verabreichung genetischen materials
DK0681483T3 (da) 1993-01-26 2005-11-21 Univ Pennsylvania Præparater og fremgangsmåder til administration af genetisk materiale
US7001759B1 (en) 1993-01-26 2006-02-21 The Trustees Of The University Of Pennsylvania Compositions and methods for delivery of genetic material
GB9304239D0 (en) * 1993-03-01 1993-04-21 British Bio Technology Viral particles
US5858838A (en) 1998-02-23 1999-01-12 Taiwan Semiconductor Manufacturing Company, Ltd. Method for increasing DRAM capacitance via use of a roughened surface bottom capacitor plate
US6291157B1 (en) 1998-02-23 2001-09-18 Connaught Laboratories Limited Antigenically-marked non-infectious retrovirus-like particles
US5955342A (en) * 1994-08-15 1999-09-21 Connaught Laboratories Limited Non-infectious, replication-defective, self-assembling HIV-1 viral particles containing antigenic markers in the gag coding region
US6080408A (en) 1994-08-22 2000-06-27 Connaught Laboratories Limited Human immunodeficiency virus type 1 nucleic acids devoid of long terminal repeats capable of encoding for non-infectious, immunogenic, retrovirus-like particles
CN1185811A (zh) * 1995-03-31 1998-06-24 H·沃尔夫 依赖于逆转录病毒样颗粒的抗原呈递系统
US6013506A (en) * 1995-06-05 2000-01-11 Wardley; Richard C. Feline leukemia virus vaccines
US6121021A (en) * 1997-12-16 2000-09-19 Connaught Laboratories Limited Constitutive expression of non-infectious HIV-like particles
AU2596600A (en) 1998-12-31 2000-07-31 Chiron Corporation Modified hiv env polypeptides
WO2000039304A2 (fr) 1998-12-31 2000-07-06 Chiron Corporation Polynucleotides codant pour des polypeptides antigeniques du type c du vih; polypeptides et leurs utilisations
US8623379B2 (en) 2000-03-02 2014-01-07 Emory University Compositions and methods for generating an immune response
AU1194802A (en) 2000-03-02 2001-12-11 Univ Emory DNA expression vectors and methods of use
ATE445411T1 (de) 2001-03-08 2009-10-15 Us Gov Health & Human Serv Mva-exprimierende modifizierte hiv-envelope-, gag-und pol-gene
US7211659B2 (en) 2001-07-05 2007-05-01 Chiron Corporation Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US20030170614A1 (en) 2001-08-31 2003-09-11 Megede Jan Zur Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides and uses thereof
CN105709221B (zh) 2011-04-06 2020-11-17 拜欧瓦克西姆有限公司 用于预防和/或治疗人中的hiv疾病的药物组合物
WO2017015463A2 (fr) 2015-07-21 2017-01-26 Modernatx, Inc. Vaccins contre une maladie infectieuse
US11045540B2 (en) 2017-03-15 2021-06-29 Modernatx, Inc. Varicella zoster virus (VZV) vaccine
WO2018170256A1 (fr) 2017-03-15 2018-09-20 Modernatx, Inc. Vaccin contre le virus de l'herpès simplex
EP3938379A4 (fr) * 2019-03-15 2023-02-22 ModernaTX, Inc. Vaccins à base d'arn contre le vih

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0241239A2 (fr) * 1986-04-07 1987-10-14 THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce Lignée cellulaire produisant des antigènes viraux du SIDA sans produire des particules virales infectieuses
EP0381146A1 (fr) * 1989-02-02 1990-08-08 Banyu Pharmaceutical Co., Ltd. Cellules T clonées produisant de HIV antigénique et non infectieux
EP0386882A1 (fr) * 1989-02-06 1990-09-12 Dana Farber Cancer Institute Provirus d'HIV défectif pour l'empaquetage, lignées cellulaires et leur utilisation
WO1991005864A1 (fr) * 1989-10-13 1991-05-02 Connaught Laboratories Limited Production par genie genetique de vaccin contre le sida et contre les maladies retrovirales
WO1991005860A1 (fr) * 1989-10-16 1991-05-02 Whitehead Institute For Biomedical Research Particules non infectieuses de vih-1 et leurs emplois

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0241239A2 (fr) * 1986-04-07 1987-10-14 THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce Lignée cellulaire produisant des antigènes viraux du SIDA sans produire des particules virales infectieuses
EP0381146A1 (fr) * 1989-02-02 1990-08-08 Banyu Pharmaceutical Co., Ltd. Cellules T clonées produisant de HIV antigénique et non infectieux
EP0386882A1 (fr) * 1989-02-06 1990-09-12 Dana Farber Cancer Institute Provirus d'HIV défectif pour l'empaquetage, lignées cellulaires et leur utilisation
WO1991005864A1 (fr) * 1989-10-13 1991-05-02 Connaught Laboratories Limited Production par genie genetique de vaccin contre le sida et contre les maladies retrovirales
WO1991005860A1 (fr) * 1989-10-16 1991-05-02 Whitehead Institute For Biomedical Research Particules non infectieuses de vih-1 et leurs emplois

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
AIDS RES. HUM. RETROVIRUSES vol. 6, no. 1, 1990, page 63 V. KARAKOSTAS 'Assembly of retrovitus particles lacking env protein in cells infected with recombinant vaccinia virus expressing the HIV1' *
J. VIROL. vol. 63, no. 6, 1989, pages 2550 - 2556 C. PENG ET AL. 'Role of hiv type 1 specific protease' *
J. VIROL. vol. 64, no. 6, 1990, pages 2653 - 2659 O. HAFFAR ET AL. 'HIV-like nonreplicating gag-env particles assemble in a recombinant vaccinia virus expression system' *
PROC. NATL. ACAD. SCI. USA vol. 86, no. 22, 1989, pages 8964 - 8967 V. KARAKOSTAS 'HIV-like particles produced by a vaccinia virus expression vector' *
See also references of WO9107425A1 *
VIROLOGY vol. 160, no. 2, 1987, pages 323 - 329 H. GENDELMAN ET AL. 'Molecular characterisation of a polymerase mutant human HIV' *

Also Published As

Publication number Publication date
AU6905591A (en) 1991-06-13
WO1991007425A1 (fr) 1991-05-30
OA09698A (en) 1993-08-30
HU9201659D0 (en) 1992-08-28
ZA909302B (en) 1991-09-25
FI922277A0 (fi) 1992-05-19
AU636944B2 (en) 1993-05-13
ES2052478T1 (es) 1994-07-16
EP0502105A4 (en) 1993-02-24
JPH05503629A (ja) 1993-06-17
FI922277A (fi) 1992-05-19
CA2068713A1 (fr) 1991-05-21
NO921969L (no) 1992-06-26
TW216446B (fr) 1993-11-21
KR920703639A (ko) 1992-12-18
HUT60506A (en) 1992-09-28
NO921969D0 (no) 1992-05-19
GR930300005T1 (fr) 1993-04-28

Similar Documents

Publication Publication Date Title
AU636944B2 (en) Non-replicating recombinant-made retroviral particles used as antiviral agents and immunogens
JP3967374B2 (ja) 同調的インビボ遺伝子発現
Stott et al. Preliminary report: protection of cynomolgus macaques against simian immunodeficiency virus by fixed infected-cell vaccine
Rusche et al. Humoral immune response to the entire human immunodeficiency virus envelope glycoprotein made in insect cells.
Hanke et al. DNA multi-CTL epitope vaccines for HIV and Plasmodium falciparum: immunogenicity in mice
JP6165612B2 (ja) コンセンサス／先祖免疫原
US7255862B1 (en) ALVAC/FIV constructs
Griffiths et al. Hybrid human immunodeficiency virus Gag particles as an antigen carrier system: induction of cytotoxic T-cell and humoral responses by a Gag: V3 fusion
Miller et al. The gag-specific cytotoxic T lymphocytes in rhesus monkeys infected with the simian immunodeficiency virus of macaques.
Giavedoni et al. Immune response of rhesus macaques to recombinant simian immunodeficiency virus gp130 does not protect from challenge infection
Rovinski et al. Expression and characterization of genetically engineered human immunodeficiency virus-like particles containing modified envelope glycoproteins: implications for development of a cross-protective AIDS vaccine
GB2181435A (en) Vaccines and immunoassays for acquired immune deficiency syndrome
Ahmad et al. Reduced virus load in rhesus macaques immunized with recombinant gp160 and challenged with simian immunodeficiency virus
WO1996006177A2 (fr) Particules semblables a des retrovirus rendues non-infectieuses par une pluralite de mutations
Winter et al. Recombinant BCG strains expressing the SIVmac251nef gene induce proliferative and CTL responses against nef synthetic peptides in mice
JP2000506727A (ja) 組換え生ネコ免疫不全ウイルスおよびプロウイルスｄｎａワクチン
Schlienger et al. Human immunodeficiency virus type 1 major neutralizing determinant exposed on hepatitis B surface antigen particles is highly immunogenic in primates
WO1997005242A1 (fr) Nouveau clone proviral du virus de l'immunodeficience humaine de type 2 (hiv-2) a possibilite de replication, appele hiv-2kr
WO2008005929A2 (fr) Immunogène vih-1 gp120 recombinant avec trois boucles v3 différentes issues de virus de différentes clades
CA2077753C (fr) Composition a base de gp120 purifie conservant sa conformation naturelle
VAN EENDENBURG et al. Cell-mediated immune proliferative responses to HIV-1 of chimpanzees vaccinated with different vaccinia recombinant viruses
IE904161A1 (en) Nonreplicating recombinant-made retroviral particles useful¹as anti-viral agents and as immunogens for prophylaxis and¹therapy against human retroviruses
Mills et al. Vaccine-induced CD4+ T cells against the simian immunodeficiency virus gag protein. Epitope specificity and relevance to protective immunity.
Schlienger et al. Vaccine-induced neutralizing antibodies directed in part to the simian immunodeficiency virus (SIV) V2 domain were unable to protect rhesus monkeys from SIV experimental challenge
WO1999019501A1 (fr) Vecteur d'origine retrovirale d'adn non integrant a forte expression de proteines et antigenes immunogenes secretes

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19920515

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LI LU NL SE

RIN1 Information on inventor provided before grant (corrected)

Inventor name: TRAVIS, BRUCE, M.

Inventor name: SENEAR, ALLEN, W.

Inventor name: HU, SHIU-LOK

Inventor name: HAFFAR, OMAR, K.

ITCL It: translation for ep claims filed

Representative=s name: JACOBACCI CASETTA & PERANI S.P.A.

EL Fr: translation of claims filed
TCNL Nl: translation of patent claims filed
TCAT At: translation of patent claims filed
A4 Supplementary search report drawn up and despatched

Effective date: 19930105

AK Designated contracting states

Kind code of ref document: A4

Designated state(s): AT BE CH DE DK ES FR GB GR IT LI LU NL SE

REG Reference to a national code

Ref country code: ES

Ref legal event code: BA2A

Ref document number: 2052478

Country of ref document: ES

Kind code of ref document: T1

17Q First examination report despatched

Effective date: 19970213

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19990612