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
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