Highlights of HIV Drug Resistance Studies Presented at the 3rd IAS Conference

By Ian Frank, MD

Table of Contents
	Epidemiology
	NRTI Resistance
	NNRTI Resistance
	Protease Inhibitor Resistance

Epidemiology     Top

Contacts of the New York City “Superinfection” Case Identified

Early in 2005 a New York City man was identified as an individual with apparent recent HIV infection who had multiple class resistant virus, dual-tropic virus, and experienced rapid disease progression (Markowitz et al. Lancet 2005;365:1031).

Two possible contacts, who were themselves sexual partners of this NYC case, were identified independently by Quest Diagnostics and LabCorp after searching their genotyping databases for matches to the unusual mutational pattern of the virus from the NYC case, and traced to an HIV practice in Connecticut (Blick et al. Abstract MoOa0101).    

These two contacts had a history of unprotected insertive intercourse among themselves (phylogenetic analyses of isolates suggests that one individual may have acquired the particular resistance profile of the other by superinfection), and admitted to unprotected intercourse with the New York City case at a sex club. 

Although neither of these individuals experienced rapid disease progression and both had R5, rather than mixtures of R5 and X4 phenotypes or dual tropic virus, the epidemiologic evidence for transmission of this uniquely resistant virus is strong.

The take-home messages are that 1) the community must continue to be educated that safer sex practices are important among infected individuals, 2) the HIV uninfected community should not misconstrue the benefits of today’s antiretroviral therapy to believe that becoming HIV infected means just taking a few pills a day, and 3) your genotype may be able to identify you as easily as your social security number.

Infection with Resistant Virus Is Not Associated with More Rapid Disease Progression

Motivated by the New York City case, a group of European investigators evaluated whether infection with resistant virus was associated with more rapid disease progression (Wensing et al. Abstract WeOaLB0101).

A case control study was performed using a prospective, multicenter cohort of 1415 individuals diagnosed in 2003.  78 individuals were identified at presentation with at least one primary resistance associated mutation, based upon the IAS-USA resistance algorithm.  These cases were compared to 77 randomly selected controls matched by baseline viral load and CD4+ count with sensitive virus. 

Median baseline viral loads and CD4+ counts were 4.8 log₁₀ copies/mL and 359 cells/mm³ in the cases (resistant virus) and 4.7 log₁₀ copies/mL and 365 cells/mm³ in the controls.  Subjects were followed for a median of 16 months for the development of one of three endpoints, fall in CD4+ count to <200 cells/mm³, new AIDS-defining clinical illness, or initiation of therapy. 

During follow-up, 17 subjects with resistant virus started therapy, two experienced CD4+ cell count declines to <200 and one developed an AIDS-defining event, compared to 28 subjects with sensitive virus who started therapy, one had a CD4+ count decline to <200, and two developed an AIDS-defining event.  There are no statistically significant differences in immunologic or clinical disease progression between the two groups.

These data suggest that individuals who get infected with resistant virus have similar disease progression as those who get infected with sensitive virus.

NRTI Resistance    Top

A New Mechanisms for NRTI Resistance: Mutations the RNase H Encoding Region

To date the number of RNase H sequences available in the GenBank database are relatively few, and the commercially available genotyping resistance assays do not amplify the RNase H domain (amino acids 441 – 560).  For this reason, the influence of RNase H on susceptibility to NRTIs has not been carefully evaluated.

In vitro, site directed mutants in RNase H increased the IC₅₀ value of wild type virus to ZDV and d4T by 10- to 100-fold.  These mutations conferred synergistic levels of resistance when combined with thymidine analog mutations (TAMs). 

To evaluate whether mutations in RNase H contribute to RT resistance in patients, RNase H sequences from NRTI-experienced and naïve subjects were cloned and sequenced, and then inserted into a wild RT genome, replacing the WT RNase, which could be used to test the phenotypic susceptibility of recombinant isolates. 

Among patients with RT sequences that were wild type for RT mutations in the pol gene associated with resistance, treatment experienced patients had RNase sequences that were associated with 2.4- to 5.7-fold resistance to ZDV and 1.0- to 1.8-fold resistance to d4T, in some cases, clinically significant levels of resistant. 

In contrast, the RNase sequences from treatment naïve patients had no impact on phenotypic susceptibility, suggesting that treatment may have selected for virus with RNase sequences that impart some phenotypic resistance independent of that associated with mutations in other areas of the pol gene. 

The level of phenotypic resistance was amplified in treatment experienced patients with TAMs.  The RNase H sequence from one subject with TAMs conferred 1839-fold resistance, compared to 11-fold resistance with a wild type RNase sequence.

The mechanism by which RNase H mutations cause RT resistance is not known, but is hypothesize to occur by delaying RT processivity, thereby increasing the time that excision of the terminal nucleotide could occur. More studies need to be done to investigate whether RNase mutations may be associated with resistance to non-thymidine analogs and what mutations in RNase H may be clinically significant, to ultimately determine whether genotyping and phenotyping assays need to be designed to include an evaluation of RNase H.

Absence of K65R in Subjects Failing Tenofovir in GS934

GS934 was an open-label comparison of tenofovir (Viread), emtricitabine (Emtriva), and efavirenz (Sustiva) versus fixed dose zidovudine/lamivudine and efavirenz in antiretroviral naïve subjects. Subjects who received tenofovir + emtricitabine had better virologic outcomes in the intent to treat analysis.  In Rio data on the mutational analysis in subjects with virologic failure was presented  (Pozniak et al. Abstract WeOa0202), and is summarized in the accompanying table.  Resistance in GS934*

*Excluding subjects with resistant virus at baseline 

Mutations were not identified in every subject, some of whom rebounded with wild type virus.  Asexpected, the majority of subjects with resistant virus had NNRTI resistance.  A greater proportion of subjects developed I84V/I on 3TC than FTC, though those numbers are not statistically significant.  In contrast to GS903, a comparison of TDF, 3TC (Epivir), and EFV versus d4T, 3TC, and EFV, in which 8 subjects of 47 subjects (17%) with virologic failure in the TDF arm had virus with a K65R mutation (one subject in the d4T arm developed K65R) (Gallant et al. JAMA 292; 2004;  191-201), no subjects in GS934 failing on tenofovir developed K65R.  Whether the long half-life of FTC can “protect” against the selection of K65R will be determined with data from larger studies currently underway.

The Effect of the M184V Mutation on HIV Replication and the Maintenance of CD4+ Counts

Unfortunately, many patients with quantifiable viral loads on antiretroviral therapy harbor virus that is resistant to multiple classes of drugs. In many, there is little chance that modifying therapy will reduce viral loads below quantifiable levels without the addition of two new classes of agents. 

The only option is to continue the suboptimal combination, but toxicities may make that difficult.  The M184V mutation associated with lamivudine (3TC) and emtricitabine (FTC) resistance reduces the replicative capacity of virus, and it’s been argued that maintaining the M184V mutation in the face of virologic failure may help keep viral loads in check. 

To answer this question Castagna and his Italian colleagues designed the E-184V study to determine if there was a virologic, immunologic, and clinical advantage to this strategy (Castagana Abstract WeFo0204).

In this study patients failing antiretroviral therapy with CD4+ counts of >500 cells/mm³ who had the M184V mutation were randomized to interrupt their entire combination or stop all drugs except for 3TC 300 mg QD.  Subjects resumed therapy if their CD4+ count declined to <350 cells/mm³, or if they developed HIV-related clinical signs or symptoms.

Fifty-eight subjects were included in the study with a mean CD4+ count of 566 cells/mm³ and viral load of 3.7 log₁₀ copies/mL.  They had been on therapy for a mean of 7 years and most had 3-drug class experience.  After 48 weeks of follow-up, more patients required re-initiation of therapy in the treatment interruption group than in the 3TC monotherapy group (20/29, 69% vs 12/29, 41%, p=0.018). 

No patient in the 3TC group experienced any clinical events, but their were 6 clinical events in the treatment interruption group, including acute retroviral syndrome, pneumonia, lymphoma, and candida esophagitis.  The viral load in the 3TC group remained approximately 0.5 log₁₀ copies/mL below that in the treatment interruption group (Figure 1), and although there was no statistically significant differences in the CD4+ counts between the two groups (Figure 2), the CD4+ percentage was maintained in the 3TC group compared to the treatment interruption group (Figure 3). 

For those who required re-initiation of therapy, 70% of subjects achieved a viral load <50 copies/mL on a new combination in the 3TC group compared to only 37% in the treatment interruption group.

Results from this study are the clearest demonstration to date that maintaining the M184V mutation in the face of virologic failure is a valuable strategy, and is preferable to the discontinuation of the entire combination.  

NNRTI Resistance    Top

Preventing Mother-to-Child Transmission (MTCT) and Nevirapine Resistance

A number of studies have documented the high rate of NNRTI resistance in pregnant women given single dose nevirapine (Viramune) to prevent HIV transmission, as well as the poorer response to an NNRTI-inclusive antiretroviral combination in women previously treated with single dose nevirapine. 

The Treatment Options Preservation Study (TOPS) was designed to determine whether short courses of fixed dose zidovudine plus lamivudine (ZDV/3TC) can prevent the selection of NNRTI resistance when co-administered with single dose nevirapine to prevent MTCT (McIntyre et al. Abstract TuFo0204).

All women were given a single dose of nevirapine (200 mg) during labor and their newborns were given a single dose (2 mg/kg) with 48 hours of delivery.  Women and their newborns were randomized (1:1:1) as a pair to receive no other medication or ZDV/3TC BID for four or seven days beginning during labor and within 24 hours of birth.  Population based genotypes were performed testing for NNRTI- and 3TC-resistance on mothers two and six weeks after delivery and on the newborns a birth and ages two and six weeks. 

The trial was designed to include 300 women, but was modified after 226 mothers delivered 228 infants because of a higher rate of NNRTI resistance in the single dose nevirapine alone group (see Table1).  The median CD4+ count in these 226 women was 314 cells/mm³ and the median viral load was 4.49 log₁₀ copies/mL.  The median viral load was higher in those women who developed NNRTI resistance (43,650 copies/mL in women who developed resistance compared to 10,600 whose virus remained wild type. 

At 6 weeks, the overall transmission rate was 10.5% (Gray et al. Abstract TuPe5.4P01). Two infants infected in utero had resistance at birth, in the single dose nevirapine group  and one in the sd NVP + ZDV/3TC x 4 days group.  Among newborns acquiring infection at delivery, NNRTI resistance was seen in 6 of 9 who received single dose nevirapine, 0 of 6 who received sd NVP + ZDV/3TC x 4 days, and 0 of 7 who received sd NVP + ZDV/3TC x 7 days.

The trial is continuing to accrue subjects to compare outcomes in women who receive sd NVP + ZDV/3TC x 4 versus 7 days.  In addition, women and children are being evaluated for the selection of 3TC resistance.  This trial will hopefully end the use of single dose NVP for the prevention of MTCT. 

Although these results may seem less relevant in the developed world, the results may be applicable to individuals stopping antiretroviral therapy.  These data suggest that patients on an NNRTI based combination who wish or need to stop therapy should discontinue their NNRTI for a period of time before stopping their NRTIs.  

Protease Inhibitor Resistance     Top

Establishing the Clinical Cutoffs for Atazanavir and Tipranavir

Rick Pesano from ViroLogic provided data describing the establishment of the clinical cutoffs for the protease inhibitors (PI) atazanavir (Reyataz) and tipranavir (Aptivus) relevant to their Phenosense assay (Pesano. Abstract MoFo0301).

The clinical cutoff for unboosted atazanavir was derived from specimens available from the BMS 043 trial, a comparison of unboosted atazanavir versus fixed dose lopinavir/ritonavir in patients with previous virologic failure on a PI-containing regimen; and the clinical cutoff for boosted atazanavir was derived from specimens available from BMS 045, a comparison of boosted atazanavir versus fixed-dose lopinavir/ritonavir. 

The clinical cutoff for unboosted atazanavir is 2.2 (corresponding to approximately 3 mutations), and for boosted atazanavir it is 5.2 (corresponding to approximately 5 mutations).   The clinical cutoff for boosted tipranavir is 4.0, corresponding to approximately 5 mutations. 

09/09/05

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