Molecular cloning of HIV-1 from immortalized cell lines
Inventors
Gallo, Robert C. • Wong-Staal, Flossie • Popovic, Mikulas • Hahn, Beatrice H. • Shaw, George M. • Fisher, Amanda G.
Assignees
US Department of Health and Human Services
Publication Number
US-9309574-B1
Publication Date
2016-04-12
Expiration Date
2033-04-12
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Abstract
Disclosed is the molecular cloning of HTLV-III, the adult leukemia and acquired immune deficiency syndrome (AIDS) virus. Clone BH10 contains a 9.0 Kb viral insert constituting the entire HTLV-III genome. Clones BH8 and BH5 contain viral inserts of 5.5 Kb and 3.5 Kb, respectively. These clones are suitable for the development of diagnostic and therapeutic measures for AIDS, as well as use as probes for the detection of AIDS. By scientific convention, HTLV-III, referred to herein also as HIV, has been renamed as HIV-1.
Core Innovation
The invention relates to the molecular cloning of Human Immunodeficiency Virus Type-1 (HIV-1), also known as HTLV-III, the causative agent of AIDS. The invention provides clones containing essentially the entire HIV genome, including clones BH10, BH5, and BH8, which contain viral inserts of 9.0 Kb, 3.5 Kb, and 5.5 Kb, respectively. These clones enable the reliable production of HIV viral particles and proteins in immortalized CD-4 positive cell lines, offering a dependable source of virus for research and the preparation of diagnostic and immunogenic products.
The problem addressed by the invention arises from the highly cytopathic nature of HIV, which hinders its reproduction and cloning in vitro, particularly in immortalized cell lines. Prior art had not disclosed how to clone such a highly cytopathic and genomically diverse virus. The invention overcomes this by utilizing infected immortalized cell lines to extract unintegrated and integrated HIV DNA, thereby enabling the molecular cloning of the virus.
The invention provides infectious HIV clones that can be propagated in bacteria allowing amplification of the viral genome. It also enables the production of homogeneous viral particles for vaccine evaluation and diagnostic tool development. The clones are suitable for generating specific viral proteins in eukaryotic and prokaryotic systems, offering reliable sources for immunogens and diagnostic agents. Furthermore, the clones enable the study of HIV variants and viral transmission by serving as probes to detect viral sequences in patient-derived strains.
Claims Coverage
The patent includes multiple independent claims covering compositions and methods related to specific HIV-1 nucleic acid sequences derived from molecular clones and their uses.
Composition comprising duplexes of HIV-1 nucleic acid and single-stranded nucleic acid probes
Compositions comprising duplexes formed between HIV-1 nucleic acids and single-stranded nucleic acids of at least 18 contiguous nucleotides selected from sequences of lambda bacteriophages λ-HXB2, λ-HXB3, clones BH10, BH5, BH8, pHXB3, pHXB-2D, E. coli clone X10-1, or the H9/HTLV-III cell line. The single-stranded nucleic acids do not form duplexes with HTLV-I or HTLV-II under specified stringency conditions. The duplexes exist outside mammalian cells and viral particles and feature longer single-stranded regions flanking a double-stranded region. The single-stranded nucleic acid may be labeled and may include DNA, RNA, or cDNA.
Methods for preparing DNA constructs specific for HIV-1
Methods involving insertion of nucleic acids of at least 18 contiguous nucleotides selected from the identified HIV-1 clones into vectors to prepare DNA constructs. These constructs allow RNA transcript production, DNA replication, and expression of recombinant HIV-1 polypeptides in host cells.
Single-stranded and double-stranded nucleic acids for HIV-1 detection and analysis
Single- and double-stranded nucleic acids of specified lengths derived from the disclosed HIV-1 clones and comprising sequences from gag, pol, env open reading frames, or long terminal repeat regions. These nucleic acids are used for nucleic acid hybridization with conditions discriminating HIV-1 from HTLV variants, are covalently attached to solid supports, may include detectable labels, and can be chemically synthesized or randomly generated.
Compositions for hybridization assays
Compositions comprising duplexes formed between single-stranded nucleic acids from the specified HIV-1 clones and HIV-1 nucleic acids encoding full-length gag, pol, env polypeptides, or long terminal repeat regions. These duplexes are used in hybridization under discriminating stringency conditions, may be bound to solid supports, and may include compounds such as sodium saline citrate, formamide, or dextran sulfate.
The independent claims collectively cover compositions of nucleic acid duplexes formed between HIV-1 nucleic acid sequences derived from molecular clones and complementary single-stranded probes, methods for constructing and replicating HIV-1 DNA constructs, and compositions suitable for nucleic acid hybridization assays with high specificity distinguishing HIV-1 from related viruses.
Stated Advantages
Provides a reliable source of HIV, viral particles, proteins, and antibodies via clones containing essentially the entire HIV genome.
Enables production of specific viral proteins in eukaryotic and prokaryotic systems for diagnostic evaluation and vaccine development.
Supplies homogeneous viral particles useful for vaccine evaluation and study of variant HIV strains.
Enables detection of HIV viral sequences in patient samples for diagnosis and study of disease transmission.
Allows propagation and amplification of infectious clones in bacteria for further molecular studies.
Documented Applications
Use as probes to detect HIV viral sequences in HIV strains isolated from patients for diagnostic and research purposes.
Production of viral proteins and viral particles for development of diagnostic kits such as immunoblotting and immunoadsorbent tests.
Generation of monoclonal antibodies reactive with HIV antigens for research and diagnostic uses.
Application of viral antigens as immunogens in vaccine development against HIV.
Use of compositions comprising HIV nucleic acids hybridized to specific probes for distinguishing HIV-1 from related retroviruses HTLV-I and HTLV-II in biological samples.
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