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Resistance to Anti-HIV Medications

HIV Treatment Series: Part Two of Four

November/December 2002


Also see parts one, three and four.

How Do HIV Meds Work in the Body?

When HIV multiplies inside an infected cell, it relies on proteins called enzymes. Whenever you see a word that ends with "-ase" you're probably looking at an enzyme. First, HIV uses reverse transcriptase to read its own genetic code and copy it. This code is a set of instructions for building a new virus. Next, HIV uses integrase to insert a copy of its code into the infected cell's own "code book" in the cell nucleus. Now the virus can use the cell's own machinery to make copies of itself. New viral proteins get manufactured based on the genetic code. Then HIV uses protease to assemble these proteins into a new working copy of itself.

Our current anti-HIV drugs don't kill HIV. In fact, scientists haven't figured out how to kill any virus yet. Instead, they design drugs that make it harder for the virus to multiply. So far, the drugs we have to fight HIV block, or inhibit, a specific enzyme. Two types of drug -- the nucleoside analog reverse transcriptase inhibitors (also called nukes), and the non-nucleoside reverse transcriptase inhibitors (non-nukes or NNRTIs) -- block the reverse transcriptase enzyme. A third type of drug, protease inhibitors, blocks the final step of viral assembly that depends on the protease enzyme.

The HIV genetic code is like a string of beads called nucleosides. There are just four different nucleosides, like four colors of beads, that get put together in different combinations. The viral nucleosides are adenosine (A), guanosine (G), thymidine (T) and cytidine (C). It takes three nucleosides to define a specific amino acid. Amino acids are the basic building blocks of all life. When HIV -- or a human cell -- multiplies, the genetic code gets "read." Amino acids are created according to the genetic code and assembled into proteins to make a new cell, or a new virus.

Nucleoside Analog Reverse Transcriptase Inhibitors (NRTIs)

When reverse transcriptase (RT) reads HIV's genetic code, it goes one nucleoside or "bead" at a time. It identifies which nucleoside it is (which color) and it looks around inside the cell for that nucleoside's "mate." Each nucleoside pairs off with just one other type of nucleoside. "A" pairs up with "T," and "G" pairs off with "C." When the RT enzyme reads an "A," it looks for a "T" and vice versa. When it reads a "G," it looks for a "C" and vice versa.

The nucleoside analog RT inhibitors are fake nucleosides ("analog" means "something similar"). The enzyme can't tell the difference and picks up a drug molecule instead of a real nucleoside. The fake nucleosides stop the process of reverse transcription. It's kind of like a zipper, where reverse transcriptase is the "pull" that combines the two sides. One side is HIV's genetic code, and the other side is made up of the nucleosides that reverse transcriptase finds inside the cell. A "nuke" drug molecule is like a bent tooth on the zipper, and the process can't continue.

Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs)

Researchers tell us that the RT enzyme is shaped kind of like a catcher's mitt. The strand of HIV genetic code slides between the "thumb" and the "fingers" of the enzyme, along the palm of the hand. The NNRTI drugs block the RT enzyme by filling up the pocket in the catcher's mitt. When there's a baseball in the mitt, nothing can slide along the "palm." The enzyme can't read the genetic code.

Protease Inhibitors (PIs)

HIV uses the protease enzyme late in its life cycle. After all the new viral proteins have been built, they get assembled into a new virus that takes shape and pushes out of the infected cell. The insides of this new virus aren't fully formed yet, and protease plays a key role. It's like a pair of chemical scissors that cut long strands of protein into the correct pieces needed to assemble the core of the virus. The protease inhibitor drugs block the enzyme by locking in between the two blades of the scissors so they can't cut anything.

What Is Resistance?

If reverse transcriptase or protease got blocked 100%, HIV couldn't make any new copies of itself and your viral load would eventually decline to zero. Unfortunately, anti-HIV drugs aren't 100% effective and HIV continues to multiply. Sometimes, HIV develops resistance to a drug, or in other words, it keeps on multiplying just like the drug wasn't even there in the first place.

Resistance is a happy accident from the virus' point of view. When new viral copies are made, they almost always contain errors: slight differences in the genetic code that result in slightly different versions of HIV. Most of these errors, or mutations, are fatal to the virus and they die without multiplying. But sometimes, mutant versions of HIV can not only multiply, but can ignore some antiviral drugs.

For the nukes, we talked about a zipper getting stuck when it hit a bent tooth. With some particular mutations, HIV acts like a self-repairing zipper. Reverse transcriptase becomes able to read "around" the bent tooth -- the nucleoside analog drug molecule -- and continue creating the code for a new virus.

For the non-nukes, if the reverse transcriptase enzyme changes shape just a little bit, it might "drop the ball" that we put in the catcher's mitt. The drug molecule might not be able to stay inside the enzyme to block it, and RT can go ahead and read the viral genetic code.

For the protease inhibitors, if protease changes shape just the right way, the drug molecule might not be able to stick in between the blades of the scissors. Protease can go ahead and do its job of assembling a new virus.

Not every mutation can give HIV resistance to drugs. It's a random process. The more new copies HIV makes, the more mutations there are, and the more likely it is that some of them will give the virus resistance to antiviral drugs.

Why Does Resistance Matter?

If your virus develops resistance to a medication, it will keep multiplying even though you take the drug. Resistance can cut down your possible treatment options very quickly. For example, the latest version of the treatment guidelines lists 78 different antiviral combinations as "strongly recommended" or "recommended alternatives." But if your virus develops resistance to AZT (Retrovir), for example, and you can't use it any more, then there are only 39 remaining combinations you could choose from.

AZT and d4T (Zerit), both from the NRTI class, are somewhat cross-resistant. If your virus becomes highly resistant to AZT, it's probably at least partly resistant to d4T. If you can't use either AZT or d4T in your regimens, then there are only 13 combinations to choose from.

The current guidelines don't include tenofovir (Viread), which was recently approved. That will add several more options for people who can't use AZT or d4T. The important point is, however, you don't want to run out of options and have to wait for a new medication to get approved.

What Can I Do About Resistance?

When HIV multiplies, it mutates. That's just a fact of viral life. Once in a while, one of those mutations will help the virus resist medications. The more HIV multiplies, the more it mutates, and the higher the risk of new resistance mutations showing up. The best way to avoid resistance is to keep it from developing in the first place. And the best way to do that is to keep HIV under control so it has fewer chances to multiply.

To keep HIV from multiplying, you should take anti-HIV medications according to their instructions. When you do this, you should have enough of each drug in your bloodstream to keep HIV under control. The manufacturers work hard to figure out how much drug is needed to control the virus without causing too many side effects.

If you miss doses or if you don't take them with (or without) food, according to the instructions, there might not be enough drug in your bloodstream. When drug levels drop, HIV can multiply more quickly, at least for a while. More multiplication of HIV means more mutations and a higher risk of developing resistance.

There have been some studies showing that resistance develops even at undetectable levels. Others show it can occur even with 100% adherence. Researchers are searching for the mechanisms that allow this to happen. In the meantime, what you can control is your adherence (as long as you can tolerate your regimen). Research shows that in the era of HAART (highly active antiretroviral therapy), your first shot is your best shot. If you have a regimen that's working, be sure to stick to it!

Testing for Resistance

There are two main ways to test HIV for resistance to drugs: genotypic and phenotypic testing. Both types of test use a blood sample. In most cases, the patient should have a viral load of at least 1,000 for the tests to work properly.

Genotypic testing examines the genetic code of the virus and looks for mutations. That is, it looks for changes from the normal (or "wild type") sequence of nucleosides in the genes that contain the instructions for the reverse transcriptase and the protease enzymes. Over the years, researchers have studied strains of HIV that are resistant to each of the anti-HIV drugs. They analyzed the genetic sequences of the resistant virus and defined the specific mutations that seemed to always show up in resistant virus. Genotypic testing looks for these mutations.

Researchers developed a code to identify specific mutations. Since it takes three nucleosides to define a specific amino acid, they counted them in groups of three (called "codons") along the gene for either reverse transcriptase or protease. For example, a particular mutation at codon number 184 of the reverse transcriptase gene can give HIV resistance to the drug 3TC (Epivir). This mutation replaces the wild type (normal) amino acid, which is Methionine, with a different amino acid: Valine. In research shorthand, this mutation is the "M184V" mutation: instead of Methionine at codon 184, there is Valine. With this one mutation, HIV has a high level of resistance to Epivir.

A single mutation can also give HIV resistance to all of the NNRTIs: the K103N mutation. Just like the previous example, "K" and "N" are codes for amino acids. Instead of the genetic code for "K" at position 103 of reverse transcriptase, we find the code for amino acid "N". When the same mutation or group of mutations makes HIV resistant to more than one drug, those drugs are called "cross-resistant." For example, the PIs Crixivan (indinavir) and Norvir (ritonavir) are cross-resistant. All of the NNRTIs are cross-resistant.

It's not always this clear whether HIV has resistance to a certain drug or not. For most of the protease inhibitor drugs, HIV has to get several mutations, one after another, before it develops resistance.

The resistance test report for genotypic testing is a list of mutations found in the sample of HIV. Those mutations that are believed to cause resistance to specific drugs are highlighted, and the report usually indicates whether the virus is believed to be resistant or sensitive to each anti-HIV drug. Unfortunately, when the virus needs multiple mutations to develop resistance, it's not always clear whether it's resistant or not. Genotypic testing cannot tell you "how resistant" the virus is to any particular drug, but resistance is not an all-or-nothing thing. HIV can be sensitive to a drug (no resistance), slightly resistant (the drug still works, but not as well as against wild type virus), or highly resistant (the drug doesn't slow HIV down at all). In some cases, mutations can make the virus hypersusceptible: a drug might work even better than against the wild type virus.

Phenotypic Testing is the second main type of resistance testing. Instead of the genetic code of the virus, it looks at how fast the virus actually multiplies when each anti-HIV drug is present. A range of doses of each drug is added to individual test tubes containing cultures of either the sample virus or of "wild type" virus. After a certain amount of time, the amount of the sample virus in each test tube is measured and compared to the amount of "wild type" virus. If there are more copies of the sample virus than the wild type virus, it has resistance to the drug.

Resistance is reported as "fold change" in a phenotypic test report. This tells you how many times more copies there are of the sample virus compared to the wild type virus. For example, if there are 500,000 copies of the wild type virus, and 2 million copies of the sample virus, then it has "4-fold" resistance to the drug being studied. Although this is easy to understand, it's not clear what it means. For some drugs, 4-fold resistance means that the drug won't work at all. For other drugs, 10-fold resistance means that the virus is still sensitive to the drug. There are "cut-off" levels for fold resistance used in phenotypic resistance reports. They are different for each drug, and are somewhat different for each company.

Which Test Is Better?

Genotypic testing is indirect. It analyzes the genetic code of the virus, and reports on mutations that researchers have found to be related to resistance to particular drugs. It can be difficult to decide whether a certain collection of mutations means the virus is resistant or not. Genotypic testing has to be interpreted using a set of rules, and each company doing resistance testing might use a slightly different set of rules for its reports. Also, the rules keep changing as researchers learn more about exactly which combinations of mutations are the most relevant to resistance for each drug. Genotypic testing is faster (about one week) and less expensive than phenotypic testing.

Phenotypic testing is a direct measure of how the virus behaves in the presence of anti-HIV drugs. The report is easy to understand: it tells you how much "fold resistance" the sample virus has to each drug. However, it can be hard to know what level of fold resistance really matters. Phenotypic testing usually takes about two weeks and is more expensive than genotypic testing.

New Types of Resistance Tests

The company Tibotec-Virco provides the "Virtual Phenotype" resistance test. It's priced between genotypic and phenotypic tests and the results come back faster than phenotypic testing. First the virtual phenotype does a genotypic test. Then, instead of using a set of rules to interpret the list of mutations found in the sample, the results are compared to a large database of paired genotypic and phenotypic test results. The test report tells you the phenotypic test results of samples in the database with similar mutation patterns.

ViroLogic provides the "PhenoSense GT" test. It's not really a different type of test. However, for doctors who prefer to see both genotypic and phenotypic test results, it uses a single blood sample to run both tests and provides the test results in a side-by-side format.

Weaknesses of Resistance Testing

There are other problems with resistance testing besides the difficulties in interpreting results.

  • Neither test can detect "minority" strains that make up less than about 20% of a patient's population of viruses.
  • They cannot detect resistance that might be "hiding" in resting T-cells or other viral reservoirs, and some researchers believe that this "archived" resistance can re-emerge quickly if it will help the virus survive particular drugs.
  • When someone stops taking antiviral medications, the drug-resistant virus has no survival advantage, and the wild type virus is likely to re-emerge and become the most common strain. The tests may not detect any resistance if the patient has been off medications for more than a couple of weeks.
  • The rules for interpreting genotypic tests, and the fold change results for phenotypic tests are all based on just one drug at a time and may not accurately reflect what's going to happen with a combination of anti-HIV meds.
  • Some doctors use both genotypic and phenotypic tests to get a more complete picture of what's going on, but often the tests give conflicting results that can be very confusing to interpret.

    Does Resistance Testing Help?

    Several clinical trials studied whether doctors who had the results of resistance tests made better treatment decisions for their patients. The doctors just made their treatment decisions the normal way (without resistance test results), or they got genotypic and/or phenotypic test results. Sometimes they got expert advice on how to interpret the resistance test results.

    In most of the trials, resistance test results led to viral loads about .5 log lower than those without. That's a significant difference. Unfortunately, not every trial showed the same results, and in some cases it was difficult to tell whether it was resistance test results or expert advice that made the most difference. Although there are still some challenges in using resistance test results, most AIDS physicians believe that they help make better treatment decisions.

    Who Should Get a Resistance Test?

    Treatment guidelines recommend resistance testing in the following situations:

  • When antiviral treatment stops working: if viral load rises rapidly or CD4 cell count drops.
  • When antiviral treatment isn't working well enough: viral load doesn't become undetectable within a month or two.
  • Pregnant HIV-positive women.

    Guidelines suggest that resistance testing be "considered" for newly-infected people. Several research studies have documented an increasing rate of new infections with strains of HIV that are already resistant to one or more antiviral drugs. Resistance testing might help doctors avoid prescribing drugs that won't control a patient's virus. Using the wrong drugs could allow HIV to multiply and develop additional resistance.

    Probably the most controversial in terms of resistance testing are people not taking antiviral medications who have been infected for several months or more. As mentioned above, if you're not taking antiviral medications, the wild type virus will usually multiply the fastest and become the dominant strain in your body. However, some researchers believe that certain mutations can persist and be detected for several months or maybe even longer. There hasn't been a lot of research on this question yet.

    The take home message is to prevent resistance in the first place. The best way to avoid developing resistance is to take your anti-HIV medicines on schedule and as according to instructions. If you have a regimen that is working for you, then stick to it!

    For additional discussion on drug resistance also see "Once Again, One a Day."


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    See Also
    The Body's Guide to HIV Drug Resistance
    More on Drug Resistance