No abstract

Even in the late era of highly effective antiretroviral treatments, detection of CWR, particularly if extended to all three drug classes is related to poorer clinical outcome and represents a risk-marker of disease progression and death.

Amino acid conservation in HIV PR can be minimally present in 45 residues out of 99. Identification of these invariable residues, with crucial roles in dimer stability, protein flexibility and catalytic activity, and their mapping on the three-dimensional structure of the enzyme will help guide the design of novel resistance-evading drugs.

To define the extent of sequence conservation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in vivo, the first 320 amino acids of RT obtained from 2,236 plasma-derived samples from a well-defined cohort of 1,704 HIV-1-infected individuals (457 drug naïve and 1,247 drug treated) were analyzed and examined in structural terms. In naïve patients, 233 out of these 320 residues (73%) were conserved (<1% variability). The majority of invariant amino acids clustered into defined regions comprising between 5 and 29 consecutive residues. Of the nine longest invariant regions identified, some contained residues and domains critical for enzyme stability and function. In patients treated with RT inhibitors, despite profound drug pressure and the appearance of mutations primarily associated with resistance, 202 amino acids (63%) remained highly conserved and appeared mostly distributed in regions of variable length. This finding suggests that participation of consecutive residues in structural domains is strictly required for cooperative functions and sustainability of HIV-1 RT activity. Besides confirming the conservation of amino acids that are already known to be important for catalytic activity, stability of the heterodimer interface, and/or primer/template binding, the other 62 new invariable residues are now identified and mapped onto the three-dimensional structure of the enzyme. This new knowledge could be of help in the structure-based design of novel resistanceevading drugs.

In patients failing cART with LLV, HIV-1 genotyping provides reliable and reproducible results that are informative about emerging drug resistance.

Background: We tested whether pre-HAART viraemia affects the achievement and maintenance of virological success in HIV-1-infected patients starting modern firstline therapies. Methods: A total of 1,430 patients starting their first HAART (genotype-tailored) in 2008 (median; IQR: 2006-2009) were grouped according to levels of pre-HAART viraemia (≤30,000, 30,001-100,000, 100,001-300,000, 300,001-500,000 and >500,000 copies/ml). The impact of pre-therapy viraemia on the time to virological success (viraemia ≤50 copies/ml) and on the time to virological rebound (first of two consecutive viraemia values >50 copies/ml after virological success) were evaluated by Kaplan-Meier curves and Cox regression analyses. Results: Median pre-HAART viraemia was 5.1 log 10 copies/ml (IQR 4.5-5.5), and 53% of patients had viraemia >100,000 copies/ml. By week 48, the prevalence of patients reaching virological success was >90% in all pre-HAART viraemia ranges, with the only exception of range >500,000 copies/ml (virological success =83%; P<0.001). Higher pre-HAART viraemia was tightly correlated with longer median time to achieve virological success. Cox multivariable estimates confirmed this result: patients with pre-HAART viraemia >500,000 copies/ml showed the lowest hazard of virological undetectability after adjusting for age, gender, pre-HAART CD4 + T-cell count, transmitted drug resistance, calendar year and third drug administered (adjusted hazard ratio [95% CI]: 0.27 [0.21, 0.35]; P<0.001). Pre-HAART viraemia >500,000 copies/ml was also associated with higher probability of virological rebound compared with patients belonging to lower viraemia strata at weeks 4, 12 and 24 (P=0.050). Conclusions: At the time of modern HAART, and even though an average >90% of virological success, high pre-HAART viraemia remains an independent factor associated with delayed and decreased virological success. Patients starting HAART with >500,000 copies/ml represent a significant population that may deserve special attention.HAART has significantly extended the time to development of AIDS and to death in HIV-infected individuals [1,2]. Its efficacy in suppression of plasma HIV-1 RNA to undetectable levels, and in increasing CD4 + T-cell count, is well documented in several clinical trials [3][4][5][6].Despite years of great progress in treating AIDS, however, in some patients starting their first treatment; the effectiveness of HAART is still not sufficient, with consequent virological failures [7][8][9]. These failures can be caused by several factors, such as drug potency, drug

Plasma-derived sequences of human immunodeficiency virus type 1 (HIV-1) protease from 1,162 patients (457 drug-naïve patients and 705 patients receiving protease inhibitor [PI]-containing antiretroviral regimens) led to the identification and characterization of 17 novel protease mutations potentially associated with resistance to PIs. Fourteen mutations were positively associated with PIs and significantly correlated in pairs and/or clusters with known PI resistance mutations, suggesting their contribution to PI resistance. In particular, E34Q, K43T, and K55R, which were associated with lopinavir treatment, correlated with mutations associated with lopinavir resistance (E34Q with either L33F or F53L, or K43T with I54A) or clustered with multi-PI resistance mutations (K43T with V82A and I54V or V82A, V32I, and I47V, or K55R with V82A, I54V, and M46I). On the other hand, C95F, which was associated with treatment with saquinavir and indinavir, was highly expressed in clusters with either L90M and I93L or V82A and G48V. K45R and K20T, which were associated with nelfinavir treatment, were specifically associated with D30N and N88D and with L90M, respectively. Structural analysis showed that several correlated positions were within 8 Å of each other, confirming the role of the local environment for interactions among mutations. We also identified three protease mutations (T12A, L63Q, and H69N) whose frequencies significantly decreased in PI-treated patients compared with that in drug-naïve patients. They never showed positive correlations with PI resistance mutations; if anything, H69N showed a negative correlation with the compensatory mutations M36I and L10I. These mutations may prevent the appearance of PI resistance mutations, thus increasing the genetic barrier to PI resistance. Overall, our study contributes to a better definition of protease mutational patterns that regulate PI resistance and strongly suggests that other (novel) mutations beyond those currently known to confer resistance should be taken into account to better predict resistance to antiretroviral drugs.

Most commercial HIV-1 genotyping assays are hampered by high cost in resource-limited settings. Moreover, their performance might be influenced over time by HIV genetic heterogeneity and evolution. An in-house genotyping protocol was developed, and its sequencing performance and reproducibility were compared to that of ViroSeq TM . One hundred ninety plasma samples from HIV-1-infected subjects in Cameroon, a resource-limited setting with a high HIV genetic variability, were processed for pol gene sequencing with an in-house protocol, ViroSeq TM , or both. Only non-B subtypes were found. The in-house sequencing performance was 98.7% against 92.1% with ViroSeq TM . Among 36 sequence pairs obtained using both assays, the overall rate of discordant amino acid positions was negligible (0.24%). With its high sensitivity and reproducibility, as well as its affordable cost (about half of ViroSeq TM : 92 € vs. 217 €), this in-house assay is a suitable alternative for HIV-1 genotyping in resource-limited and/or in high-genetic-diversity settings.