Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2001-06-25
2004-10-12
Fredman, Jeffrey (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S091200, C536S024300, C536S024320
Reexamination Certificate
active
06803187
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention relates to the field of HIV diagnosis. More particularly, the present invention relates to the field of diagnosing the susceptibility of an HIV sample to antiviral drugs used to treat HIV infection.
The present invention relates to a method for the rapid and reliable detection of drug-selected mutations in the HIV protease gene allowing the simultaneous characterization of a range of codons involved in drug resistance using specific sets of probes optimized to function together in a reverse-hybridization assay.
2. BACKGROUND OF THE INVENTION
The human immunodeficiency virus (HIV) is the ethiological agent for the acquired immunodeficiency syndrome (AIDS). HIV, like other retroviruses, encodes an aspartic protease that mediates the maturation of the newly produced viral particle by cleaving viral polypeptides into their functional forms (Hunter et al). The HIV protease is a dimeric molecule consisting of two identical subunits each contributing a catalytic aspartic residue (Navia et al, Whodawer et al, Meek et al). Inhibition of this enzyme gives rise to noninfectious viral particles that cannot establish new cycles of viral replication (Kohl et al, Peng et al).
Attempts to develop inhibitors of HIV-1 protease were initially based on designing peptide compounds that mimicked the natural substrate. The availability of the 3-dimensional structure of the enzyme have more recently allowed the rational design of protease inhibitors (PI) using computer modeling (Huff et al, Whodawer et al). A number of second generation PI that are partially peptidic or entirely nonpeptidic have proven to exhibit particularly potent antiviral effects in cell culture. Combinations of various protease inhibitors with nucleoside and non-nucleoside RT inhibitors have also been studied extensively in vitro. In every instance, the combinations have been at least additive and usually synergistic.
In spite of the antiviral potency of many recently developed HIV-1 PI, the emergence of virus variants with decreased sensitivity to these compounds has been described both in cell culture and in treated patients thereby escaping the inhibitory effect of the antiviral (Condra et al.). Emergence of resistant variants depends on the selective pressure applied to the viral population. In the case of a relatively ineffective drug, selective pressure is low because replication of both wild-type virus and any variants can continue. If a more effective drug suppresses replication of virus except for a resistant variant, then that variant will be selected. Virus variants that arise from selection by PI carry several distinct mutations in the protease coding sequence that appear to emerge sequentially. A number of these cluster near the active site of the enzyme while others are found at distant sites. This suggests conformational adaptation to primary changes in the active site and in this respect certain mutations that increase resistance to PI also decrease protease activity and virus replication.
Amongst the PI, the antiviral activity of the PI ritonavir (A-75925; ABT-538). nelfinavir (AG-1343), indinavir (MK-639; L735; L524) and saquinavir (Ro 31-8959) have been approved by the Food and Drug Administration and are currently under evaluation in clinical trials involving HIV-infected patients. The VX-487 (141W94) antiviral compound is not yet approved. The most important mutations selected for the above compounds and leading to gradually increasing resistance are found at amino acid (aa) positions 30 (D to N), 46 (M to I), 48 (G to V), 50 (I to V), 54 (I to A, I to V), 82 (V to A, or F, or I, or T), 84 (I to V) and 90 (L to M). Other mutations associated with drug resistance to the mentioned compounds have been described (Schinazi et al). Saquinavir-resistant variants, which usually carry mutations at amino acid positions 90 and/or 48, emerge in approximately 45% of patients after 1 year of monotherapy. Resistance appears to develop less frequently with higher doses of saquinavir. Resistance to indinavir and ritonavir requires multiple mutations; usually at greater than 3 and up to 11 sites, with more amino acid substitutions conferring higher levels of resistance. Resistant isolates usually carry mutations at codons 82, 84, or 90. In the case of ritonavir, the mutation at codon 82 appears fist in most patients. Although mutant virions resistant to saquinavir are not cross-resistant to indinavir or ritonavir, isolates resistant to indinavir are generally ritonavir resistant and visa versa. Resistance to either indinavir or ritonavir usually results in cross-resistance to saquinavir. Approximately one third of indinavir resistant isolates are cross-resistant to nelfinavir as well.
The regime for an efficient antiviral treatment is currently not clear at all. Patterns of reduced susceptibility to HIV protease inhibitors have been investigated in vitro by cultivating virus in the presence of PI. These data, however, do not completely predict the pattern of amino-acid changes actually seen in patients receiving PI. Knowledge of the resistance and cross-resistance patterns should facilitate selection of optimal drug combinations and selection of sequences with non-overlapping resistance patterns. This would delay the emergence of cross-resistant viral strains and prolong the duration of effective antiretroviral activity in patients. Therefore, there is need for methods and systems that detect these mutational events in order to give a better insight into the mechanisms of HIV resistance. Further, there is need for methods and systems which can provide data important for the antiviral therapy to follow in a more time-efficient and economical manner compared to the conventional cell-culture selection techniques.
3. AIMS OF THE INVENTION
It is an aim of the present invention to develop a rapid and reliable detection method for determination of the antiviral drug resistance of viruses, which contain protease genes such as HIV retroviruses present in a biological sample.
More particularly it is an aim of the present invention to provide a genotyping assay allowing the detection of the different HIV protease gene wild type and mutation codons involved in the antiviral resistance in one single experiment.
It is also an aim of the present invention to provide an HIV protease genotyping assay or method which allows to infer the nucleotide sequence at codons of interest and/or the amino acids at the codons of interest and/or the antiviral drug selected spectrum, and possibly also infer the HIV type or subtype isolate involved.
Even more particularly it is an aim of the present invention to provide a genotyping assay allowing the detection of the different HIV protease gene polymorphisms representing wild-type and mutation codons in one single experimental setup.
It is another aim of the present invention to select particular probes able to discriminate wild-type HIV protease sequences from mutated or polymorphic HIV protease sequences conferring resistance to one or more antiviral drugs, such as ritonavir (A-75925; ABT-538), nelfinavir (AG-1343), indinavir (MK-639; L735; L524), saquinavir (Ro 31-8959) and VX-478 (141W94) or others (Shinazi et al).
It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV protease sequences from mutated or polymorphic HIV protease sequences conferring resistance to ritonavir (A-75925; ABT-538).
It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV protease sequences from mutated HIV protease sequences conferring resistance to nelfinavir (AG-1343).
It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV protease sequences from mutated HIV protease sequences conferring resistance to indinavir (MK-639; L735; L524).
It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV protease sequences from mutated HIV prote
Fredman Jeffrey
Howrey Simon Arnold & White , LLP
Innogenetics N.V.
Switzer Juliet C.
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