Download Article PDF/SlidesResistance of HIV to antiretroviral drugs is one of the most common causes for therapeutic failure in people infected with HIV. Sadly, the emergence of drug-resistant HIV variants is a common occurrence—even under the best of circumstances—given that no antiretroviral drug combination studied as of yet is completely effective in shutting down viral replication. And there is no shortage of data indicating that the emergence of HIV drug resistance is clearly associated with adverse treatment outcomes.
Fortunately, the availability of drug-resistance testing has improved the ability of clinicians to deal knowledgeably with HIV drug resistance head on. On the research front, drug-resistance testing has enabled investigators to more effectively develop and study both novel and older therapeutics for the sake of tailoring treatment for patients with varying resistance profiles. In this respect, therapy can now be individualized, based on our evolving knowledge of drug resistance, drug-resistance testing, and state-of-the-art treatment approaches.
But if clinicians are to fully appreciate the epidemiology, prognostic tests, and various treatment choices related to HIV drug resistance, it is important to understand the mechanisms by which HIV drug resistance evolves. To explain this, and to illustrate some of the recent advances elucidating the mechanisms of HIV drug resistance, Dr. François Clavel took the podium at PRN’s annual holiday dinner in December to provide a basic and not-so-basic overview.
| Selection of Drug-Resistant Mutants | Top of page |
To understand how mutations associated with drug resistance arise, it is necessary to start at the beginning. At the time of infection, a single virus may be introduced and goes on to replicate with the help of a single CD4+ cell. A number of new virions are produced, each of which goes on to use other CD4+ cells to replicate further. During this process, even in its earliest stages, different strains of the virus are produced. These strains differ from one another by random mutations in their genetic structures. Some of these mutations are minor and are called base substitutions or amino acid substitutions. Other mutations are more significant, as they involve combinations of amino acid substitutions, deletions, or insertions.
“Some of these mutations are good for the virus, as they can help the virus to escape the pressure of the immune system, providing it with a survival advantage,” Dr. Clavel explained. “Other mutations are harmful to the virus. In fact, most mutations are harmful to HIV, as they can cause the virus to create stops or changes in proteins that are essential for replication. In turn, these species of virus quickly disappear and are overgrown by strains that have better replicative capacity. But overall, as time progresses, there is constant diversification of HIV.”
With the start of a single antiretroviral agent, it is likely that treatment will be initially effective in reducing the dominant, usually “wild type,” strain of HIV. However, among the diverse population of virus, there will likely be at least one strain harboring a particular mutation that confers a small survival advantage in the presence of the particular antiretroviral drug. If this variant strain is permitted to continue replicating, it will continue to diversify, with some of progeny virus accumulating additional mutations that may confer greater resistance to the antiretroviral agent being used. Eventually, a variant will likely emerge that harbors enough key mutations to fully resist the agent being used, thereby rendering it the dominant and uncontested dominant strain. “If we use a second drug, to combat virus that has become resistant to the first drug, the process repeats itself,” Dr. Clavel said.
This is usually slow process, as it can take numerous rounds of replication and competition among the diversified strains to render one variant that has a strong survival advantage compared to other variants in the presence of the antiretroviral agent being used. But in some cases, high-level drug resistance can be almost instantaneous. This is certainly the situation with nevirapine (Viramune) and lamivudine (Epivir). Upon initiating either of these two drugs as monotherapy, high-level resistance emerges within days to weeks because of the proliferation of variants containing the K103N and M184V mutations, respectively. With other drugs, such as the protease inhibitors and nucleoside analogues, several mutations are necessary and, as a result, take longer to confer high-level resistance.
With the advent of combination antiretroviral therapy, it was believed that the selection of drug-resistant mutations could be halted or slowed considerably. First, Dr. Clavel explained, “it’s very unlikely that you will find a variant in patients who have never been treated that is resistant to all three drugs being used in a combination. It’s just basic statistics. It’s statistically impossible to find such a virus. Second, because a combination of drugs is more powerful than single-agent therapy, we end up blocking the capacity of any breakthrough virus to acquire additional mutations that can lead to high-level resistance seen in the sequential monotherapy scenario.”
Unfortunately, no drug combination studied to date has been shown to completely shut down viral replication. In turn, the virus continues to diversify and eventually accumulate enough key mutations to pose a challenge to the antiretroviral drug regimen being used. “This tends to be a slow process,” Dr. Clavel added. “It may begin with resistance to one drug and subsequent diversification that leads to resistance to the second drug. And with resistance to two drugs complete, we’re looking at the monotherapy scenario, which can quickly translate into resistance to all three drugs being used. However, the virus has an easier time resisting some medications than others when used in combinations. Resistance to lamivudine usually occurs quickly and is often the first evidence of resistance to emerge. Resistance to stavudine [Zerit] is much more complex and can take much longer. Resistance to the protease inhibitors falls somewhere in between.”
To understand this, Dr. Clavel contends that it is necessary to explore the methods of resistance that occur in each of the classes of currently available antiretroviral drugs.
| The Selective Advantage of Protease Mutants | Top of page |
Virus resistance is usually calculated by measuring the concentration of drug that is required to inhibit 50% (IC50) of virus infectivity. For each HIV variant, the level of resistance is calculated relative to the virus’s infectivity in drug-free conditions. Dr Clavel and his group have measured resistance in a slightly different way. Rather than calculating mutant virus inhibition relative to drug-free conditions, they have measured replication of mutant relative to wild-type virus over a range of drug concentrations. This amounts to measuring the selective advantage of that mutant as a function of the concentration of drug, thus defining a unique and characteristic "fitness profile." The fitness profiles that were calculated for viruses representing each of the mutational pathways studied were fully consistent with the observations made in vivo regarding the order of appearance of the mutations in treated patients. By integrating the main parameters of the selection for drug resistance, drug-free infectivity, resistance, and drug concentration, Dr. Clavel argues that it is possible to map out the pattern of accumulation of protease inhibitor-associated resistance mutations.
To conduct this exploration, Dr. Clavel’s group reconstructed a large series of HIV mutants carrying single or combined protease mutations, retracing the pathways of resistance observed in patients treated with ritonavir (Norvir), saquinavir (Invirase), indinavir (Crixivan), and nelfinavir (Viracept). First taking a look at ritonavir resistance, Dr. Clavel’s group found that most of the mutations that are usually combined in ritonavir-resistant strains of HIV did not confer any selective advantage when present alone in the virus, no matter what concentration of the drug was used. However, there was a notable exception. The V82A mutation appeared to confer a small level of selective advantage in the presence of low ritonavir concentrations. Perhaps not coincidentally, this is usually the first mutation to emerge, in vivo, during ritonavir therapy.
With the gradual accumulation of resistance mutations, there was a notable increase in the extent and the range of the selective advantage. With ritonavir, variants harboring combinations of A71V and V82A among other mutations were the most efficient virus variant, with the maximal advantage obtained for the mutant carrying all four of the tested mutations (46I, 54V, 71V, and 82A) in combination.
The experience with saquinavir told a similar story. Only one single mutation conferred selective advantage to the virus: L90M. Interestingly, the G48V mutation, which results in significant drug resistance using standard testing, did not appear to confer a selective advantage when present alone. With continued evolution toward higher levels of saquinavir resistance, the most favorable combination of mutations—L10I, G48V, and L90M—markedly outperformed the other mutants both in the extent of the replicative performance and in the range of drug concentrations over which this advantage could be perceived.
The same held true for nelfinavir. Resistance to nelfinavir will move either along the D30N pathway or the L90M pathway. According to Dr. Clavel, mutants harboring either mutation alone did have a selective advantage, with the D30N mutation resulting in the most profound selective advantage. Neither the N88D nor the A71V mutation, alone, conferred any selective advantage to the virus. With a combination of mutations, most notably D30N and A71V, the selective advantage of the mutant virus increased substantially.
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