Cloning and expression of HTLV-III DNA
Inventors
Chang, Nancy T. • Gallo, Robert C. • Wong-Staal, Flossie
Assignees
National Institutes of Health NIH • US Department of Health and Human Services
Publication Number
US-8785609-B1
Publication Date
2014-07-22
Expiration Date
2031-07-22
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Abstract
The determination of the nucleotide sequence of HTLV-III DNA; identification, isolation and expression of HTLV-III 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 HTLV-III.
Core Innovation
The invention relates to the cloning and expression of HTLV-III DNA in recombinant vector host systems that are capable of producing immunoreactive HTLV-III polypeptides. This includes the determination of the nucleotide sequence of HTLV-III DNA, identification and isolation of HTLV-III sequences encoding immunoreactive polypeptides, and production of viral RNA. The recombinant polypeptides produced are immunoreactive with sera from AIDS patients and antibodies to HTLV-III, and are useful in immunoassays to detect HTLV-III and antibodies against HTLV-III in body fluids.
The problem being solved arises from the difficulty in diagnosing AIDS before clinical manifestations appear, the absence of preventive methods for the disease, and the high mortality rate once AIDS develops. HTLV-III is implicated as the etiological agent of AIDS, but because of its unique properties distinct from other HTLV viruses, and because viral envelope antigens like p41 are partially destroyed during virus inactivation and purification, it has been difficult to characterize HTLV-III antigens and develop sensitive diagnostic and therapeutic methods. There is a vital need for methods to detect HTLV-III or antibodies thereto, to screen blood and other body fluids, and to develop immunotherapies and vaccines to prevent and treat AIDS.
The invention provides genetic engineering methods to isolate and clone HTLV-III DNA sequences encoding immunoreactive polypeptides including gag, pol, env, sor, and Px gene regions, produce these polypeptides in recombinant systems such as E. coli, and use them to develop immunochemical assays, vaccines, and antibodies, including monoclonal antibodies. The determination of the nucleotide sequence of HTLV-III DNA enables the design of specific DNA and RNA probes that uniquely identify HTLV-III, differentiating it from HTLV-I or HTLV-II. These probes and polypeptides together provide a basis for diagnosis, prevention by screening of blood and blood components, and potential immunotherapies against AIDS.
Claims Coverage
The claims focus on methods of detecting human immunodeficiency virus (HIV) nucleic acid using specific nucleic acid probes. There are multiple inventive features including probe specificity, hybridization conditions, and probe composition.
Use of HIV-specific nucleic acid probes for detection
Methods involving contacting a fluid or bodily fluid sample suspected of containing HIV nucleic acid with nucleic acid probes comprising nucleotide sequences complementary to or identical to the nucleotide sequence depicted in FIG. 3, or portions thereof, to specifically hybridize to HIV nucleic acid if present.
Discrimination from related viruses
Nucleic acid probes and methods employed are specific such that they form a duplex with HIV nucleic acid but do not form a duplex with HTLV-I or HTLV-II nucleic acids, allowing specific detection of HIV distinct from related retroviruses.
Probe sequences from specific genomic regions
The nucleic acid probes comprise sequences complementary to or identical with the gag, pol, env-lor open reading frames, or LTR regions of HIV as delineated in FIG. 3. Specific nucleotide ranges within these regions are recited in the claims, enabling targeted detection.
RNA or DNA probe composition and labeling
The probes used in these methods can be RNA or DNA molecules, and may be labeled to facilitate detection of hybridization.
The claims collectively cover methods of detecting HIV nucleic acid with high specificity using nucleic acid probes derived from sequences disclosed in FIG. 3 of the patent, ensuring discrimination from related viruses HTLV-I and HTLV-II, and encompassing diverse probe compositions including RNA, DNA, and labeled forms.
Stated Advantages
Provides sensitive and specific detection of HTLV-III (HIV) and antibodies against it, enabling early diagnosis of AIDS.
Facilitates screening of blood and blood components to prevent transmission of AIDS.
Allows production of immunoreactive polypeptides and monoclonal antibodies to improve immunoassay accuracy and reduce false positives.
Enables development of immunotherapeutic methods including vaccines and passive immunization against AIDS.
Documented Applications
Diagnosis of AIDS by detecting HTLV-III or antibodies against it in body fluids such as blood, saliva, and semen.
Screening donated blood and blood components (e.g., Factor VIII for hemophilia treatment) to prevent transmission of AIDS.
Immunoassays based on recombinant HTLV-III polypeptides and monoclonal antibodies for detection of virus presence.
Production of vaccines employing immunogenic HTLV-III polypeptides to elicit neutralizing antibodies for AIDS prevention.
Passive immunotherapy using antibodies against HTLV-III polypeptides.
Use of DNA and RNA probes specific to HTLV-III for detecting the virus in body fluids, enabling faster and easier testing especially in saliva.
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