Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2000-12-14
2003-03-04
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S252100, C435S320100, C440S012610, C514S04400A, C536S023100
Reexamination Certificate
active
06528251
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method of managing the chemotherapy of patients who are HIV positive, as well as a clinical management device for use by physicians treating such patients based on the phenotypic drug sensitivity of human HIV strains for inhibitors of one or more enzymes of the pol gene of HIV, as well as a method for simultaneously determining the phenotypic drug sensitivity of two or more of the enzymes of the pol gene of HIV to inhibitors thereof.
BACKGROUND ART
To date, several chemotherapeutic regimens have been developed for treating HIV infected patients. Certain of these regimens have been approved for clinical use, and others are the subject of on-going clinical trials. It can be assumed that the number of approved chemotherapeutic regimens will increase steadily in the near future. Increasingly, combination therapy or multiple drug treatment regimens are being used because of the development of drug-resistant HIV variants during therapy. Although these chemotherapeutic regimens have been shown to exert an effect on virological (viral load), immunological and clinical parameters of HIV disease, practical experience teaches that these effects are transient. In particular, one finds that the HIV strains infecting an individual patient after a while start to display reduced sensitivity to the drug or drug combination with which said patient is being treated. The loss of efficacy of the chemotherapy can vary from patient to patient, from drug to drug, or from drug combination to drug combination. It is well established that the loss of efficacy to a particular type of chemotherapy can be associated with a genotypic pattern of amino acid changes in the genome of the HIV strains infecting the patient. This probably renders these HIV strains less susceptible to the chemotherapy. As an HIV infected patient is exposed to several chemotherapeutic regimens over extended periods of time, more complex patterns of amino acid changes in the genome of infecting HIV strains occur which for the present defeat a rational approach to the further treatment of the infected patient. As implied in the previous explanation, one can routinely determine the genotypic changes occurring in HIV strains exposed to different chemotherapeutic regimens involving single or multiple anti-HIV drugs, but thus far it has proven very difficult to derive from these data information enabling a physician in charge of prescribing the chemotherapy whether or not it is sensible to initiate or continue a particular chemotherapeutic regimen. In other words, the genotypic information which is available on a limited scale, cannot routinely be translated into phenotypic information enabling the responsible physician to make the crucial decision as to which chemotherapy a patient should preferably follow. The problem also exists for drug-naive patients who become infected by drug-resistant HIV strains.
Viral load monitoring is becoming a routine aspect of HIV care. However, viral load number alone cannot be used as a basis for deciding which drugs to use alone or in combination.
Combination therapy is becoming increasingly the chemotherapeutic regimen of choice. When a person using a combination of drugs begins to experience drug failure, it is impossible to know with certainty which of the drugs in the combination is no longer active. One cannot simply replace all of the drugs, because of the limited number of drugs currently available. Furthermore, if one replaces an entire chemotherapeutic regimen, one may discard one or more drugs which are active for that particular patient. Furthermore, it is possible for viruses which display resistance to a particular inhibitor to also display varying degrees of cross-resistance to other inhibitors.
Ideally, therefore, every time a person has a viral load test and a viral load increase is detected, a drug sensitivity/resistance test should also be carried out. Until effective curative therapy is developed, management of HIV disease will require such testing.
Currently there does exist a phenotyping method which is based on virus isolation from plasma in the presence of donor peripheral blood mononuclear cells (PBMCs), and subsequent phenotyping in said cells (Japour, A. J., et al. (1993) Antimicrobial Agents and Chemotherapy; Vol. 37, No. 5, p1095-1101). This co-cultivation method, which is advocated by the AIDS Clinical Trial Group (ACTG)—particularly for phenotyping AZT (synonymous herein with zidovudine/Retrovir (Retrovir is a Trade Mark)) resistance, is time-consuming, costly and too complex to be used on a routine basis.
A phenotypic recombinant virus assay for assessment of drug susceptibility of HIV Type 1 isolates to reverse transcriptase (RT) inhibitors has been developed by Kellam, P. and Larder, B. A. (Antimicrobial Agents and Chemotherapy (1994) Vol. 38, No. 1, p23-30). This procedure allows the generation of viable virus by homologous recombination of a PCR-derived pool of RT coding sequences into an RT-deleted, noninfectious proviral clone, pHIV&Dgr;RTBstEII. Analysis of two patients during the course of zidovudine therapy showed that this approach produced viruses which accurately exhibited the same genotype and phenotype as that of the original infected PBL DNA. However, the procedure involves isolation of the patient virus by co-cultivation of patient plasma or patient PBMCs with donor PBMCs. Such prior cultivation of virus may distort the original virus composition. Furthermore, this method, although allowing one to determine the sensitivity of the isolates to various inhibitors, does not provide the physician with information as to whether to continue with the existing chemotherapeutic regimen or to alter the therapy.
Also when one enzyme only of the pol gene is being studied, the method does not readily lend itself to routine phenotypic assessment of combination therapy which conventionally involves the use of one protease and 2 RT inhibitors.
The nested PCR (polymerase chain reaction) procedure used in ti recombinant virus assay can lead to a situation where the recombinant virus does not truly reflect the situation with the HIV strains infecting the patient under investigation. This problem resides in DNA sequence homology and the minimum amount of homology required for homologous recombination in mammalian cells (C. Rubnitz, J. and Subramini, S. (1984) Molecular and Cellular Biology Vol. 4, No. 11, p2253-2258). Accordingly, any phenotypic assay based on the recombinant virus approach should endeavour to ensure that as much as possible of the patient material is amplified and that there is maximum recombination.
Thus, the RNA extraction and nested PCR procedures employed should ensure that the viral genetic material is amplified such that the amplified material maximally reflects the viral genetic diversity in the patient being investigated.
In current clinical practice there is therefore a hard-felt need (a) to determine rapidly and on a routine basis the phenotypic drug sensitivity of HIV strains infecting a particular patient, (b) to process the thus obtained data into easily understood information, and (c) to initiate, continue or adjust on the basis of said information the chemotherapy prescribed for said particular patients.
DISCLOSURE OF THE INVENTION
According to a first aspect of the invention there is provided a method of managing HIV chemotherapy of patients who are HIV positive, which comprises transfecting a cell line susceptible to infection by HIV with a sequence from the pol gene of HIV, obtained by isolating viral RNA from a sample of a biological material from a patient and reverse transcribing the desired region of said pol gene, and a HIV-DNA construct from which said sequence has been deleted, culturing said transfected cells so as to create a stock of chimeric viruses providing an indication of the resistance profile of the circulating virus, assessing the phenotypic sensitivity of said chimeric viruses to an inhibitor of said enzyme encoded by the pol gene of HIV and assigning a value thereto constr
de Béthune Marie-Pierre
Hertogs Kurt
Pauwels Rudi
Birch Stewart Kolasch & Birch, LLP.
Horlick Kenneth R.
Kim Young
Virco N.V.
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