Cloning and expression of HIV-1 DNA
Inventors
Chang, Nancy T. • Gallo, Robert C. • Wong-Staal, Flossie
Assignees
National Institutes of Health NIH • US Department of Health and Human Services • Office of Technology Transfer
Publication Number
US-9328391-B1
Publication Date
2016-05-03
Expiration Date
2033-05-03
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Abstract
The determination of the nucleotide sequence of HIV-1 DNA; identification, isolation and expression of HIV-1 DNA sequences which encode immunoreactive polypeptides by recombinant DNA methods and production of viral RNA are disclosed. Such polypeptides can be employed in immunoassays to detect HIV-1.
Core Innovation
The invention concerns the determination of the nucleotide sequence of HIV-1 DNA (previously HTLV-III), identification, isolation, and recombinant expression of HIV-1 DNA sequences encoding immunoreactive polypeptides, as well as production of viral RNA. These polypeptides can be used in immunoassays to detect HIV-1 infection. The invention enables production of diagnostic and immunotherapeutic materials related to HIV-1.
The problem addressed is the difficulty in diagnosing AIDS before clinical manifestations emerge, the absence of preventive methods against the disease, and the lack of successful treatments. At the time, AIDS was characterized by severe immunodeficiency caused by HTLV-III (HIV-1), which had been isolated from many patients and implicated as the etiological agent. There was a critical need for diagnostic tools to detect infection early and prevent transmission, particularly through blood transfusion.
This invention solves the problem by cloning HTLV-III DNA into recombinant vectors that express immunoreactive polypeptides. These recombinant polypeptides allow detection of antibodies against HIV-1 in body fluids and can be used in immunotherapy, including vaccination and passive immunization. Additionally, the invention provides nucleotide sequences enabling the design of DNA and RNA probes specific to HIV-1 for direct viral detection in biological samples, addressing the need for improved diagnosis and disease prevention methods.
Claims Coverage
The patent discloses a single independent method claim directed to nucleic acid hybridization techniques for detecting HIV-1 nucleic acids using specific nucleotide sequences.
HIV-1 nucleic acid hybridization outside of mammalian cells or viral particles
A method forming a nucleic acid complex composed of a double-stranded region and two longer single-stranded regions, where the complex comprises HIV-1 nucleic acid from bodily fluid and a nucleic acid probe with a nucleotide sequence of HIV-1 depicted in FIG. 3 or a portion thereof. The complex is formed externally to mammalian cells and viral particles.
Specific hybridization to HIV-1 nucleic acid and not to HTLV-I or HTLV-II nucleic acids
The nucleic acid probe specifically hybridizes to complementary HIV-1 sequences and does not form complexes with HTLV-I or HTLV-II nucleic acids, ensuring specificity of the detection method.
Use of nucleic acid probes with detectable moiety not consisting of additional nucleic acids
Nucleic acid probes are covalently attached to detectable moieties that are not additional nucleic acids, facilitating direct detection of HIV-1 nucleic acids in samples.
Formation of nucleic acid complexes or duplexes bound to solid supports
Methods include forming nucleic acid duplexes between HIV-1 nucleic acids in samples and specific probes, where these complexes are bound to solid supports, enabling assay formats such as solid-phase hybridization assays.
Use of nucleic acid probe sequences corresponding to specific HIV-1 genome regions
The nucleotide sequences utilized in the probe are specifically from regions depicted in FIG. 3, notably nucleotides 3554 to 6664, corresponding to pol or env sequence regions, and include defined restriction fragments (e.g., 2.3 kb KpnI-KpnI, 1.0 kb EcoRI-EcoRI, EcoRI-BglII).
Hybridization under defined buffer conditions
The nucleic acid complex formation occurs in an environment containing compounds such as sodium citrate, polyvinylpyrrolidone, Ficoll, and bovine serum albumin to enhance specific hybridization during assay performance.
The single independent claim encompasses a method for specific hybridization-based detection of HIV-1 nucleic acids in bodily fluids, using recombinant DNA-derived nucleotide sequences as probes. The inventive features focus on specificity to HIV-1 over related viruses, formation of hybridization complexes outside of cells and viral particles, attachment to solid supports, employment of particular genomic sequences, use of probes with detectable moieties, and defined hybridization conditions to enable accurate detection in diagnostic settings.
Stated Advantages
Enable early detection of HIV-1 infection by detecting immunoreactive polypeptides and antibodies against HIV-1 in body fluids.
Provide tools for screening blood and blood components to prevent transmission of HIV-1 through transfusion.
Facilitate development of vaccines and immunotherapies including passive immunization using antibodies raised against HIV-1 polypeptides.
Improve accuracy of diagnostic assays by enabling direct detection of HIV-1 in biological fluids using specific DNA and RNA probes.
Allow production of monoclonal antibodies for enhanced diagnostic and therapeutic applications with reduced false positives.
Documented Applications
Diagnosis of AIDS by detecting antibodies against HIV-1 in body fluids such as blood, saliva, and semen.
Screening of blood and blood components prior to transfusion to prevent HIV-1 transmission.
Production of immunoassays employing recombinant HIV-1 polypeptides and monoclonal antibodies for direct detection of HIV-1 antigen.
Development of vaccines comprising immunogenic HIV-1 polypeptides to elicit neutralizing antibodies for prevention of AIDS.
Passive immunotherapy using neutralizing antibodies against HIV-1 to provide immunity to individuals.
Use of DNA and RNA probes derived from cloned HIV-1 nucleotide sequences to detect the virus in biological fluids by nucleic acid hybridization assays.
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