Stable HIV-1 integrase diversity during initial HIV-1 RNA Decay suggests complete blockade of plasma HIV-1 replication by effective raltegravir-containing salvage therapy
© Noguera-Julian et al.; licensee BioMed Central Ltd. 2013
Received: 1 October 2013
Accepted: 27 November 2013
Published: 5 December 2013
There is legitimate concern that minority drug-resistant mutants may be selected during the initial HIV-1 RNA decay phase following antiretroviral therapy initiation, thus undermining efficacy of treatment. The goal of this study was to characterize viral resistance emergence and address viral population evolution during the first phase of viral decay after treatment containing initiation.
454 sequencing was used to characterize viral genetic diversity and polymorphism composition of the HIV-1 integrase gene during the first two weeks following initiation of raltegravir-containing HAART in four ART-experienced subjects. No low-prevalence Raltegravir (RAL) drug resistance mutations (DRM) were found at baseline. All patients undergoing treatment received a fully active ART according to GSS values (GSS ≥ 3.5). No emergence of DRM after treatment initiation was detected. Longitudinal analysis showed no evidence of any other polymorphic mutation emergence or variation in viral diversity indexes.
This suggests that fully active salvage antiretroviral therapy including raltegravir achieves a complete blockade of HIV-1 replication in plasma. It is unlikely that raltegravir-resistant HIV-1 may be selected in plasma during the early HIV-1 RNA decay after treatment initiation if the administered therapy is active enough.
There is legitimate concern that minority drug-resistant mutants may be selected during the initial HIV-1 RNA decay phase following antiretroviral therapy initiation, when decreasing but still detectable HIV-1 RNA levels transiently coexist with suboptimal drug levels. Studies using allele-specific PCR suggested that drug-resistant HIV-1 might be selected and replicate in the first weeks of suppressive antiretroviral therapy (ART) [1, 2]. If they were confirmed, such observations would support using intensified ART regimens to accelerate viremia suppression and curtail drug resistance evolution, but not only in treatment-naïve subjects initiating first-line ART. Achieving fast blockade of HIV replication would even be more important in subjects with drug-resistant HIV-1 initiating salvage ART, given that they are more vulnerable to drug resistance evolution and are closer to exhausting their treatment options.
In the past, however, first-line treatment with 4, 5, or more antiretrovirals did not produce better virological outcomes than standard 3-drug ART . Indeed, evolutionary studies in macaque models  suggested that viral replication was completely shut down by therapy and that development of antiretroviral drug resistance during the initial viremia decay was unlikely to occur, provided that treatments were active enough and resistant variants did not pre-exist. If HIV replication could be shut down with standard ART, addition of further drugs to ART regimens would not provide additional protection against drug resistance.
Mathematical models of HIV resistance kinetics predict faster selection of resistant viruses with more potent regimens. On the other hand, previous studies correlated higher resistance selection with longer time to achieving undetectable HIV-1 RNA levels [1, 2]. raltegravir (RAL) achieves fast viremia suppression when combined with an optimized backbone treatment (OBT) , but has a low-genetic barrier and HIV-1 variants with single raltegravir drug resistance mutations could pre-exist at low levels [6, 7].
We thus sought to explore in a proof-of-concept study whether raltegravir-resistant mutants were selected during the initial HIV-1 RNA decay following initiation of raltegravir-containing ART in 4 treatment-experienced subjects with HIV-1 resistance to at least 2 antiretroviral drug classes using ultrasensitive, quantitative 454 sequencing. We also sought to characterize the diversity and viral population shifts in HIV-1 integrase during this initial HIV-RNA decay phase.
Subject clinical details
Date of diagnostic
Previous ART lines
#drugs before RAL
New drugs started alongside RAL
ddI + 3TC + DRV/r + RAL
TDF/FTC + DRV/r + RAL
TDF/FTC + DRV/r + RAL + T-20
DRV/r + RAL + MVC
Baseline CD4 (cells/mm 3)
The genotypic susceptibility score (GSS) of the salvage treatment was ≥3.5 in all patients according to population sequencing. Sensitivity scores were further confirmed considering 454 deep sequencing data obtained for integrase and protease/retrotranscriptase, when available (see Additional file 2: Table S1). At baseline, median (interquartile range) for HIV-1 ARN and CD4+ T cell counts were, 116.500 (57.750; 215.000) copies/mL and 399 (300;614) cells/mm3. Viral load decline at day 7, when available, was more than 1log copies/mL.
We observed stability of HIV-1 integrase diversity and heterogeneity within the first phase of HIV-1 RNA decay. The type and frequency of polymorphisms in sequential samples during early HIV-1 RNA decay suggests that salvage antiretroviral therapy including raltegravir is able to achieve an effective blockade of HIV-1 replication in plasma, even in heavily treatment-experienced individuals, in a context where a potent antiretroviral regimen, defined by a GSS ≥ 3, can be constructed. Of note, the rapid decline on HIV-1 RNA levels may also affect the capability of detecting minor polymorphisms using 454. When the number of obtained sequences is higher than the number of expected initial viral templates, re-sequencing of the same minor variants may lead to an artifactual appearance of low prevalence polymorphisms. Interestingly, minority polymorphisms were more frequent and fluctuated at later time points, at very low levels. Thus, obtained minor variant frequencies must be interpreted accounting for sampling limitations. Conversely, sampling limitations are not expected to significantly affect viral diversity due to the much larger impact of highly prevalent variants over minority ones in these measures.
Importantly, the blockade in replication observed in this study must be understood as a result of the global antiretroviral treatment pressure against the whole viral quasi-species. Indeed, additional 454 data on protease and reverse transcriptase at baseline confirms the full potency of the prescribed regimen, as GSS scores were not modified by the few minor resistant variants additionally detected. Our findings contrast with the rapid emergence of raltegravir resistance when non- fully active regimens (GSS < 3) are used [6, 7]. Also, antiretroviral drug resistance might evolve in other compartments or reservoirs with low drug penetration in the presence of residual HIV-1 replication. It is also worth noting that the sample size in this study is low (n = 4), thus reducing our ability to observe emergence of resistance, and that GSS in our study ranged from 3.5 to 4, so we cannot conclude whether a GSS = 3 would achieve similar results.
In conclusion, raltegravir-resistant HIV-1 may not be selected in plasma during the early HIV-1 RNA decay after salvage treatment initiation, provided that subjects are given a fully effective regimen that is able to shut down plasma replication. Further studies are required to assess whether similar findings might also be observed with other drugs or regimens and to clarify what is the minimum GSS (i.e. 3, 4 or higher) required to block HIV-1 evolution during HIV-1 RNA decay following ART initiation.
This study was supported through an unrestricted research grant from Merck Sharpe & Dohme, ‘CHAIN, Collaborative HIV and Anti-HIV Drug Resistance Network’, Integrated Project no. 223131, funded by the European Commission Framework 7 Program, the Spanish AIDS network ‘Red Temática Cooperativa de Investigación en SIDA’ (RD06/0006), and the ‘Gala contra la sida – Barcelona 2011’. Work Contained in this paper was presented in the 19th Conference on Retroviruses and Opportunistic Infections, March, 2012, Seattle, WA, USA.
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