Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2002-07-10
2004-09-28
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S091200, C536S023100
Reexamination Certificate
active
06797464
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to human immunodeficiency virus (HIV) detection assays, particularly to detection of drug-resistant HIV.
BACKGROUND OF THE INVENTION
Various assays have been developed to detect HIV. A common HIV-1 detection assay utilizes quantitative polymerase chain reaction (PCR) as a means to amplify and detect viral RNA present in patient plasma. HIV-1 positive individuals undergoing combination antiviral therapy (i.e., receiving two or more anti-HIV-1 compounds) can exhibit decreased viral loads in the peripheral blood. In some cases, after several weeks or months of therapy, HIV-1 RNA cannot be detected in the peripheral blood, indicating possible eradication of HIV-1 in those individuals. Unfortunately, if patients exhibiting such a seemingly negative result stop therapy in the face of continued viral replication below the sensitivity of detection assays, the HIV can rebound very rapidly. Thus, the limited sensitivity of HIV detection assays provides a challenge to further advances in therapy.
Highly active antiretroviral therapy (HAART) with reverse transcriptase and protease inhibitors markedly suppresses HIV replication in the majority of infected patients. However, in a significant percentage of patients, viruses acquire mutations in their reverse transcriptase and protease genes that confer decreased sensitivity to the reverse transcriptase and protease inhibitors. This leads to drug failure, a resumption of viral replication, and a negative impact on patient survival. To combat drug resistance, clinicians switch therapies to drug combinations not previously administered to the patient. However, drug resistance mutations can confer resistance to a broad spectrum of reverse transcriptase and protease inhibitors. More effective clinical management of patients is achieved if the drug-resistance mutations are identified early so that treatments can be adjusted to those that are effective against the particular strain (e.g., mutant) of HIV. In view of this, viral genotyping is becoming an established procedure for monitoring of patients on HAART and has a significant impact on patient survival.
Viruses bearing drug-resistance mutations cannot generally be identified unless they comprise a significant percentage of a patient's viral population. As a consequence, the drug-resistant viruses are usually identified relatively late in the process leading to drug failure. Therefore, there is an urgent need for approaches that provide early identification of drug-resistance viral variants in patients.
SUMMARY OF THE INVENTION
The invention is based on the discovery that even in patients having no virus detectable in the blood by known means, e.g., patients undergoing drug therapy, such as combination drug therapy, it is possible to assay for the presence of a drug resistant HIV by detecting 2-LTR (long terminal repeats) circles, e.g., in peripheral blood mononuclear cells (PMBCs), and examining the circles for the presence of mutations that confer drug resistance, e.g., known mutations. Thus, the invention features a new method of detecting polymorphisms that confer drug resistance in HIV infection and can be used to detect drug-resistant HIV before a subject develops symptoms typical of drug resistance. The invention features a method of detecting a drug-resistant HIV in a subject. The method includes the steps of obtaining a biological sample from a subject, isolating an HIV 2-LTR circle from the biological sample, and detecting a drug-resistance mutation in the HIV 2-LTR circle, such that the presence of a drug-resistance mutation in the HIV 2-LTR circle indicates the presence of a drug-resistant HIV in the subject. The method may further include the step of amplifying the 2-LTR circle (e.g., using the polymerase chain reaction). In some embodiments, the biological sample is from a subject (e.g., human or non-human primate) that is undergoing drug therapy that includes administering to the subject at least one HIV reverse transcriptase inhibitor, at least one HIV protease inhibitor, or at least one HIV reverse transcriptase inhibitor and at least one HIV protease inhibitor. The subject can be an HIV-1-positive mammal, e.g., anon-human primate or a human. In certain embodiments, the biological sample is a peripheral blood mononuclear cell. In some cases HIV viral RNA is not detected in the blood of the subject.
The invention also includes a method for determining a drug regime for treating a subject infected with HIV that includes the steps of obtaining a biological sample (e.g., a blood sample or peripheral blood monocytes) from the subject, isolating an HIV 2-LTR circle from the biological sample, determining whether a gene in the HIV 2-LTR circle has a drug-resistance mutation, and if a drug-resistance mutation is present in the gene, determining a drug regime for the subject such that the drug regime includes at least one drug against which the gene having a drug-resistance mutation does not confer drug resistance. The method may further include the step of amplifying the 2-LTR circle (e.g., using the polymerase chain reaction). In some embodiments, the biological sample is from a subject (e.g., a human or non-human primate) that is undergoing drug therapy that includes administering to the subject at least one HIV reverse transcriptase inhibitor, at least one HIV protease inhibitor, or at least one HIV reverse transcriptase inhibitor and at least one HIV protease inhibitor. The subject can be an HIV-1-positive mammal, e.g., a non-human primate or a human. In certain embodiments, the biological sample is a peripheral blood mononuclear cell. In some cases HIV viral RNA is not detected in the blood of the subject.
The invention can be used on a sample from any mammal that harbors or is suspected of harboring HIV including non-human primates (e.g., chimpanzees) and humans.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although suitable methods and materials for the practice or testing of the present invention are described below, other methods and materials similar or equivalent to those described herein, which are well known in the art, can also be used. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
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Bukrinsky et al. “A
Sharkey Mark
Stevenson Mario
Fish & Richardson P.C.
Fredman Jeffrey
University of Massachusetts
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