Ask the Experts - "Fitness" of Virus From Patients With Detectable HIV...
Ask the Experts - "Fitness" of Virus From Patients With Detectable HIV...
I provide HIV care in an inner-city clinic where many of the patients have substance abuse issues that interfere with their adherence to antiretroviral therapy. They are all referred for counseling, and we have an excellent case management program. Many of these patients have never reached undetectable viral loads and maintain HIV RNA in the 4 log10 copies/mL range with stable CD4+ counts from 250-700 cells/mm and have gone through several regimens. My feeling is that, although they have measurable virus, it is a less fit virus and less efficient at CD4+ cell destruction than wild-type virus, so I maintain the regimen while continuing to stress adherence (I am sure I could devise some deep salvage regimens but adherence would be no better, and I doubt it would achieve undetectable HIV RNA or benefit them clinically). Is there any way to confirm whether the virus is less fit, other than monitoring the CD4+ count?
This question touches on several unresolved issues involving virologic failure and the use of "salvage therapy." In my response, I will focus on the role of viral "fitness" in the setting of virologic failure and drug resistance.
The term viral fitness is vague and often misused. Viral fitness refers to the ability of a virus to replicate in a given environment. By definition, the resistant virus often seen in patients experiencing virologic failure has greater ability to replicate than wild-type virus, when measured in the presence of a drug. However, it is becoming increasingly clear that mutations associated with drug resistance come at some cost to the virus, and that the virus's inherent ability to replicate is diminished. To avoid confusion, the term "replicative capacity" is preferred, as it partially avoids the need to define the environment.
What is the evidence that drug resistance results in a virus that has less ability to replicate? The in vitro data supporting this concept are extensive, particularly with the nucleoside analogues and the protease inhibitors. For example, Goudsmit and colleagues noted that the zidovudine-related T215F/Y mutation is not stable in the absence of drug and that these mutations, therefore, confer a significant negative effect on viral replication.
Similar observations have been made with the M184V mutation associated with 3TC resistance. Patients experiencing virologic failure with 3TC monotherapy often have persistent partial viral suppression. Because M184V confers very high-level phenotypic resistance to 3TC, continued drug activity is unlikely to account for this partial suppression of viral replication. Rather, reduced replicative capacity is believed to be the primary cause, and this seems to be because M184V reduces the processivity of reverse transcriptase (ie, the ability of reverse transcriptase to complete forward transcription).
There is now a growing amount of in vitro data indicating that protease inhibitor resistance negatively affects viral replicative capacity. For example, Zennou and colleagues studied HIV variants from 5 patients failing long-term ritonavir or saquinavir therapy. The standard drug-resistance mutations emerged (V82A and L90M). Recombinant viruses containing viral gene sequences derived from these patients were observed to have decreased replicative capacity in vitro. One patient exhibited sustained but incomplete suppression of viral replication with a ritonavir-based regimen. Virus from this single patient had dramatic reductions in replicative capacity.
Nijhuis and colleagues performed similar studies on virus obtained from a person who failed ritonavir monotherapy. During early virologic failure, virus remained partially suppressed. By using recombinant viruses and growth competition experiments, they noted that these early failure variants had limited drug resistance and markedly reduced replicative capacity. However, over a period of several weeks, viral load returned to baseline. This finding was temporally associated with the emergence of a virus with replicative capacity that was greater than wild-type.
The questioner asks whether viral "fitness" can be tested by assay. Although no such assay has been developed for clinical use, there are several assays that are currently used in the research setting. Perhaps the "gold standard" is competitive growth experiments in which one virus is competed with a wild-type reference to see which replicates more efficiently (in the absence of drug). These assays are complex, lengthy, and expensive.
Recently, Wrin and colleagues at ViroLogic have developed a rapid assay that may eventually have clinical utility. This assay is a modification of their phenotypic drug susceptibility assay ("PhenoSense"). The assay involves several steps:
This procedure is performed in the absence of drug, thus giving some insight into the inherent replicative capacity of the virus.
The clinical relevance of reduced replicative capacity has not been well defined. Perhaps one of the earliest clinical observations suggesting a link was made in a series of patients infected by a nef-deleted variant of HIV and enrolled in the Sydney Blood Bank Cohort. When first described in 1992, these patients had limited evidence of CD4+ count decline, despite several years of infection. However, updated observations reported in 1999 noted that disease progression did occur, but only after prolonged periods and a reduced rate. It seems likely that HIV variants with reduced ability to replicate will also have a reduced ability to deplete CD4+ counts (loosely defined as the "virulence" of the virus).
Our group at San Francisco General Hospital recently evaluated the replicative capacity assay developed by Wrin and colleagues at ViroLogic. In a cross-sectional study of patients experiencing long-term virologic failure, viral load remained partially suppressed below pretherapy baseline levels. Compared with viruses obtained from untreated HIV-infected patients, viruses from these patients experiencing virologic failure had high-level phenotypic resistance and reduced replicative capacity. When these patients were followed longitudinally, viral load remained partially suppressed. This stable degree of partial viral suppression was associated with increased resistance and, in some cases, reduced replicative capacity.
The emerging data suggest that long-term virologic failure of antiretroviral therapy is associated with the emergence of virus that is highly resistant. However, viral load often remains partially suppressed below a pretherapy "set point." This continued partial viral suppression is likely due in part to the emergence of a virus with reduced replicative capacity ("fitness"). As a consequence of reduced levels of viral replication, CD4+ counts remain stable or decrease slowly.
Given these observations, what should be done about the patient presented here? Because complete viral suppression does not seem to possible, then continuing a partially effective regimen seems reasonable. Hopefully, this patient's regimen will maintain HIV in a state of reduced fitness, and durable CD4+ and clinical benefit will occur. However, such a strategy needs to be considered as temporary, because the benefit of reduced viral fitness is unlikely to be permanent.
I provide HIV care in an inner-city clinic where many of the patients have substance abuse issues that interfere with their adherence to antiretroviral therapy. They are all referred for counseling, and we have an excellent case management program. Many of these patients have never reached undetectable viral loads and maintain HIV RNA in the 4 log10 copies/mL range with stable CD4+ counts from 250-700 cells/mm and have gone through several regimens. My feeling is that, although they have measurable virus, it is a less fit virus and less efficient at CD4+ cell destruction than wild-type virus, so I maintain the regimen while continuing to stress adherence (I am sure I could devise some deep salvage regimens but adherence would be no better, and I doubt it would achieve undetectable HIV RNA or benefit them clinically). Is there any way to confirm whether the virus is less fit, other than monitoring the CD4+ count?
This question touches on several unresolved issues involving virologic failure and the use of "salvage therapy." In my response, I will focus on the role of viral "fitness" in the setting of virologic failure and drug resistance.
The term viral fitness is vague and often misused. Viral fitness refers to the ability of a virus to replicate in a given environment. By definition, the resistant virus often seen in patients experiencing virologic failure has greater ability to replicate than wild-type virus, when measured in the presence of a drug. However, it is becoming increasingly clear that mutations associated with drug resistance come at some cost to the virus, and that the virus's inherent ability to replicate is diminished. To avoid confusion, the term "replicative capacity" is preferred, as it partially avoids the need to define the environment.
What is the evidence that drug resistance results in a virus that has less ability to replicate? The in vitro data supporting this concept are extensive, particularly with the nucleoside analogues and the protease inhibitors. For example, Goudsmit and colleagues noted that the zidovudine-related T215F/Y mutation is not stable in the absence of drug and that these mutations, therefore, confer a significant negative effect on viral replication.
Similar observations have been made with the M184V mutation associated with 3TC resistance. Patients experiencing virologic failure with 3TC monotherapy often have persistent partial viral suppression. Because M184V confers very high-level phenotypic resistance to 3TC, continued drug activity is unlikely to account for this partial suppression of viral replication. Rather, reduced replicative capacity is believed to be the primary cause, and this seems to be because M184V reduces the processivity of reverse transcriptase (ie, the ability of reverse transcriptase to complete forward transcription).
There is now a growing amount of in vitro data indicating that protease inhibitor resistance negatively affects viral replicative capacity. For example, Zennou and colleagues studied HIV variants from 5 patients failing long-term ritonavir or saquinavir therapy. The standard drug-resistance mutations emerged (V82A and L90M). Recombinant viruses containing viral gene sequences derived from these patients were observed to have decreased replicative capacity in vitro. One patient exhibited sustained but incomplete suppression of viral replication with a ritonavir-based regimen. Virus from this single patient had dramatic reductions in replicative capacity.
Nijhuis and colleagues performed similar studies on virus obtained from a person who failed ritonavir monotherapy. During early virologic failure, virus remained partially suppressed. By using recombinant viruses and growth competition experiments, they noted that these early failure variants had limited drug resistance and markedly reduced replicative capacity. However, over a period of several weeks, viral load returned to baseline. This finding was temporally associated with the emergence of a virus with replicative capacity that was greater than wild-type.
The questioner asks whether viral "fitness" can be tested by assay. Although no such assay has been developed for clinical use, there are several assays that are currently used in the research setting. Perhaps the "gold standard" is competitive growth experiments in which one virus is competed with a wild-type reference to see which replicates more efficiently (in the absence of drug). These assays are complex, lengthy, and expensive.
Recently, Wrin and colleagues at ViroLogic have developed a rapid assay that may eventually have clinical utility. This assay is a modification of their phenotypic drug susceptibility assay ("PhenoSense"). The assay involves several steps:
Viral RNA is amplified by RT-PCR;
Patient-derived reverse transcriptase and protease gene sequences are placed in a viral vector that contains luciferase; this vector has a deletion in the env gene and is, therefore, unable to replicate;
The test vector is mixed with a second vector containing a complete env sequence (thus allowing one and only one round of replication to occur);
The vectors are used to transfect a cell line, and viruses containing patient-derived gene sequences are generated;
The amount of these viruses is normalized and used to infect a second cell line;
The amount of viral replication that occurs is then measured using luciferase activity (light count production).
This procedure is performed in the absence of drug, thus giving some insight into the inherent replicative capacity of the virus.
The clinical relevance of reduced replicative capacity has not been well defined. Perhaps one of the earliest clinical observations suggesting a link was made in a series of patients infected by a nef-deleted variant of HIV and enrolled in the Sydney Blood Bank Cohort. When first described in 1992, these patients had limited evidence of CD4+ count decline, despite several years of infection. However, updated observations reported in 1999 noted that disease progression did occur, but only after prolonged periods and a reduced rate. It seems likely that HIV variants with reduced ability to replicate will also have a reduced ability to deplete CD4+ counts (loosely defined as the "virulence" of the virus).
Our group at San Francisco General Hospital recently evaluated the replicative capacity assay developed by Wrin and colleagues at ViroLogic. In a cross-sectional study of patients experiencing long-term virologic failure, viral load remained partially suppressed below pretherapy baseline levels. Compared with viruses obtained from untreated HIV-infected patients, viruses from these patients experiencing virologic failure had high-level phenotypic resistance and reduced replicative capacity. When these patients were followed longitudinally, viral load remained partially suppressed. This stable degree of partial viral suppression was associated with increased resistance and, in some cases, reduced replicative capacity.
The emerging data suggest that long-term virologic failure of antiretroviral therapy is associated with the emergence of virus that is highly resistant. However, viral load often remains partially suppressed below a pretherapy "set point." This continued partial viral suppression is likely due in part to the emergence of a virus with reduced replicative capacity ("fitness"). As a consequence of reduced levels of viral replication, CD4+ counts remain stable or decrease slowly.
Given these observations, what should be done about the patient presented here? Because complete viral suppression does not seem to possible, then continuing a partially effective regimen seems reasonable. Hopefully, this patient's regimen will maintain HIV in a state of reduced fitness, and durable CD4+ and clinical benefit will occur. However, such a strategy needs to be considered as temporary, because the benefit of reduced viral fitness is unlikely to be permanent.