Stanford University HIV Drug Resistance Database - A curated public database designed to represent, store, and analyze the divergent forms of data underlying HIV drug resistance.

Primer on HIV Resistance

Last updated on 9/99
 

HIV Therapy and the Problem of Drug Resistance

The past three years have witnessed impressive advances in the treatment of human immunodeficiency virus (HIV) infection and AIDS (acquired immune deficiency syndrome). The currently available arsenal of drugs for the treatment of HIV infection includes agents that fall into three classes: the nucleoside analog reverse transcriptase (RT) inhibitors (NRTI), the nonnucleoside analog RT inhibitors (NNRTI) and the HIV protease inhibitors. The aim of all three drug classes is to inhibit viral replication. When an HIV-infected patient fails to respond, or stops responding to treatment with these therapeutic agents, viral drug resistance development is often the reason.

Drug resistance arises from mutations in the viral genome, specifically in the regions that encode the molecular targets of therapy, HIV protease and RT enzymes. HIV RT and protease mutations alter the viral enzymes in such a way that the enzyme's function is no longer inhibited by the drug, leaving the virus to replicate freely. An understanding of the genetic changes that render a particular drug ineffective is important to the development of new drugs, to designing the optimal drug combinations, and potentially even for the clinical management for individual patients.

The biology of HIV drug resistance

HIV exists extracellularly within virions in its double-stranded RNA form and intracellularly as DNA that is often integrated into the human host chromosome (proviral DNA). In an untreated person, from 103 to 106 virions/ml are present in circulating plasma, while the concentration of virus in lymph nodes is usually 2 to 3 orders of magnitude higher than in plasma. The average virus life cycle in most infected cells requires 1 to 2 days, and in some persons as many as 10 billion virions are produced daily.

HIV drug resistance results from the interplay of three factors: (1) HIV diversity, (2) HIV replication, and (3) anti-HIV drug selection pressure. Like many RNA viruses, HIV's replication is highly error prone; nearly one viral mutation occurs during each cycle of replication. Because of this high mutation rate, HIV exists within an individual as a complex mixture of genetically related but distinguishable variants often referred to as a "swarm" or "quasispecies". In this mixture, HIV strains containing many of the possible single amino acid substitutions (naturally occurring mutant strains) are likely to exist even before the administration of antiviral drug therapy.

When a prescribed anti-HIV treatment does not succeed in completely suppressing viral replication, the replicating HIV quasispecies is given the opportunity to develop new mutations. During drug therapy, those viruses that carry or develop mutations that confer drug resistance are selected for and eventually predominate. Certain mutations selected during drug therapy confer resistance on their own, while other mutations produce measurable resistance only when present in combination with other selected mutations. In addition, there are mutations selected during drug therapy that do not confer resistance at all, but instead compensate for the diminished activity associated with other drug-resistance mutations.

The duration of virus suppression experienced by patients receiving drug therapy depends on the time it takes for the virus population within a patient to acquire a sufficient number of drug-resistance mutations to render the therapy ineffective. This duration depends on the levels of pre-existing resistant virus strains, the number of viral mutations required to overcome the anti-HIV activity of a drug or drug combination (or the "genetic barrier" to resistance), and the fitness (rate of replication) of drug-resistant strains.

Clinical implications

The two principle laboratory parameters used to monitor disease progression and response to therapy are HIV RNA levels in plasma (the cell-free fraction of blood) and a CD4+ lymphocyte count. The concentration of HIV RNA in plasma is an excellent measurement of the rate of viral replication and is assessed with the use of quantitative gene amplification assays (e.g. polymerase chain reaction (PCR)).

Decisions on the timing and mode of treatment of HIV patients are based on three factors: (1) the patient's level of immunodeficiency as indicated by the CD4 count and whether the patient has experienced opportunistic infections; (2) the rate at which further immune system damage will occur (best determined by the plasma HIV RNA level); and (3) how enthusiastic a patient is about starting, and most importantly, adhering to a combination drug regimen. Response to treatment is assessed by periodic measurement of plasma HIV RNA levels.

Given that drug resistance evolves through a process of replication, mutation, and selection, the goal of a prescribed treatment regimen is to achieve maximal suppression of viral replication. If plasma HIV RNA levels become undetectable (<20 copies/ml of plasma) and remain undetectable for several months it is possible that HIV replication has been completely suppressed. Under these conditions, drug resistance is prevented and it is likely that complete virus suppression will be maintained for as long as treatment is continued. However, if plasma HIV RNA is persistently detectable 6 months after starting drug therapy or if plasma HIV RNA increases to detectable levels after an initial decline, then the treatment regimen is not sufficiently inhibiting HIV replication. A progressive increase in virus levels is then likely to occur as the replicating virus population acquires new drug resistance mutations.

In the treatment of most serious infections, the results of drug susceptibility testing are relied upon extensively to guide the choice of antimicrobial therapy. Although HIV is a life-threatening infection and drug resistance is a serious obstacle to successful treatment, drug susceptibility is not yet used in the routine management of HIV infection. There are at least 3 major explanations for this. First, because HIV is a virus and one that poses a hazard in the workplace, susceptibility testing has not achieved the same level as standardization than that achieved for other pathogens such as bacteria, tuberculosis, and even some less hazardous viruses (e.g. cytomegalovirus). Second, HIV's existence as a quasispecies, complicates all methods of drug susceptibility testing because minor populations of drug- resistant virus may go undetected. Finally, the usefulness of drug susceptibility testing depends on the availability of alternative treatments for patients with drug resistant virus. Although many different anti-HIV drugs are now available, the need for multiple new drugs in a treatment regimen and the problem of cross-resistance within the 3 classes of drugs limits the options for patients with drug-resistant virus.

Two types of resistance analyses are available: genotypic and phenotypic susceptibility testing. Phenotypic susceptibility testing assesses the ability of specific drugs or drug combinations to inhibit viral replication in cultured cells. Genotypic testing assesses the genetic composition of HIV variants in infected individuals to determine precisely which resistance mutations they carry. Because the currently available drugs target the HIV protease and RT proteins, their encoding nucleic acid sequences are determined in order to discover whether mutations known to render the virus resistant to a particular drug are present.

Genotypic and phenotypic testing have been used extensively in clinical drug trials, where preexisting resistance is a confounding variable that influences the effectiveness of the drug regimen under investigation. Standardized assay protocols and reagents are also available commercially and are often used in routine clinical settings. An increasing number of recent studies has shown that susceptibility testing is useful for understanding why a patient is not responding to antiviral drug therapy. For example, the presence of numerous drug-resistance mutations in an HIV isolate obtained from a patient not responding to anti-HIV therapy would suggest that drug resistance is the cause of poor response. In contrast, the absence of drug resistance mutations in an HIV isolate from a patient not responding to treatment suggests that poor adherence, decreased absorption, or rapid drug metabolism might account for treatment failure.

Unfortunately the predictive information of resistance tests is often difficult to use in putting together a better treatment regimen for a patient. A minimum of three drugs from two of the three drug classes are usually needed to achieve prolonged HIV suppression. Patients developing virologic rebound on an initial HAART regimen will often have viruses having some degree of resistance to more than one drug class. Therefore it is likely that some of the drugs in almost all salvage regimens will already be partially compromised at the start of salvage therapy. The patterns of cross-resistance within each drug class are poorly defined because most genotypic and phenotypic data on clinical HIV-1 isolates are not publicly available. Nonetheless cross-resistance is significantly greater than that indicated by the mutation lists or "look-up" tables that are familiar to most patients and physicians.

Identifying drug-resistance mutations

Drug-resistance mutations have traditionally been identified during the pre-clinical and initial clinical evaluation of a new antiretroviral drug. The pre-clinical evaluation involves culturing a wildtype HIV isolate in the presence of increasing concentrations of the drug being studied. This process selects for the development of virus mutations that enable the originally wildtype virus to replicate in the presence of high drug concentrations. The genetic sequence of the cultured virus is determined and compared to the sequence of the original HIV strain. Site-directed mutagenesis experiments are done to confirm that the mutations developing during virus passage in the presence of drug do confer drug resistance when introduced directly into a wildtype virus. Likewise, viruses that are cultured from patients during "phase I" or dose-finding studies of a new drug are sequenced and tested for resistance. The effects of mutations developing in these "clinical isolates" are also assessed in site-directed mutagenesis experiments.

Drug resistance mutations identified by this process acquire widespread acceptance as the predominant mutations responsible for resistance to the drug under evaluation, and are referred to as "canonical" resistance mutations. While the process of characterizing resistance mutations by susceptibility testing and site-directed mutagenesis is the most rigorous means of demonstrating that a particular mutation confers drug resistance, there are several limitations with this approach. In many patients, particularly those receiving combination therapy, numerous different combinations of mutations develop in drug-resistant isolates. In addition, the phenotypic effects of known drug-resistance mutations can be affected by other "background" changes in the RT or protease gene.

Once a drug has been approved by the FDA, there is less incentive to engage in studies designed to identify new mechanisms of drug resistance. Because it is costly and labor intensive, site-directed mutagenesis is not usually performed to discern the effects of mutations that arise in complicated patterns in HIV isolates from patients receiving combination therapy. Therefore many of the mutations that occur during the post-marketing use of the drug are never identified as drug-resistance mutations.

One of the hypotheses underlying this web site is that drug-resistant clinical isolates represent experiments of nature, which if analyzed correctly can complement the data compiled from in vitro studies. This database allows correlations to be made between certain drug treatments and the corresponding mutations in HIV strains isolated from patients receiving that treatment. This approach can help to identify mutant isolates that should be subjected to detailed in vitro studies. For example, if previously unrecognized mutations or collection of mutations appear to be associated with a particular drug regimen, then susceptibility testing of patient isolates carrying these mutations and the appropriate site-directed mutagenesis experiments will become a high priority.

The need for a public HIV RT and protease sequence database

Because many biological and clinical questions require analyses on ample collections of sequence data, a database that contains large numbers of representative HIV RT and protease sequences is a critical resource for researchers. Sequences of global isolates are needed to identify naturally occurring polymorphisms of HIV RT and protease. Sequences from patients treated with different anti-HIV drugs and drug combinations are needed to identify the spectrum of genetic changes selected by drug therapy. A reference database also allows researchers to rapidly compare all new HIV RT and protease sequences to those in the database. When unusual sequence variations are identified, previously reported HIV isolates with the same sequence variations can be examined.

HIV RT and protease genes have been sequenced more often than the genes of any other virus. Yet there are two major problems facing HIV researchers who study the genetic variation of these molecular targets of antiretroviral therapy. First, only a small proportion of the HIV RT and protease sequence data is in the public domain. Second, the sequences that are available publicly are not accessible in a form that is suitable for further analyses. The HIV and RT Database web site is built around a curated database that integrates genetic sequence data with detailed data about each sequence obtained from the medical literature.

GenBank, the repository of all published sequence data, contains sequences of >100,000 different genes and by necessity its annotation is highly generic. It is usually impossible to determine whether a given sequence was obtained before or after antiviral therapy or whether a set of sequences was obtained from different individuals or from one individual at different times. Queries about specific drug treatments, specific mutations, specific HIV isolates, or specific sequencing approaches are not possible. In addition, GenBank relies on passive reporting and does not include sequences that appear in journals in the form of amino acid sequence data. In contrast, a specialized database can provide curation and annotation, and attract the submission of new sequences. For example, many HIV sequences from patients receiving certain common treatments are not publicly available. This database can highlight these important gaps attracting the submission of these important sets of sequence data.

Last update: 9/23/99