The degree of HIV-1 amino acid variability is strictly related to different disease progression rates.

The aim of this study is to evaluate the amino acid variability of HIV-1 Gp41, C2-V3, and Nef in a group of patients characterized by different disease progression rates. HIV-1 sequences were collected from 19 Long term non progressor patients (LTNPs), 9 slow progressors (SPs), and 11 rapid progressors (RPs). Phylogenetic trees were estimated by MEGA 6. Differences in amino acid variability among sequences belonging to the 3 groups have been evaluated by amino acid divergence, Shannon entropy analysis, and the number of amino acid mutations (defined as amino acid variations compared with HxB2). The involvement of amino acid mutations on epitope rich regions was also investigated. The population was mainly composed of males (74.3%) and HIV-1 subtype B strains (B: 92.32%, CRF_12BF, A1, C: 2.56% each). Viral load (log10 copies/mL) and CD4+T cell count (cells/mm3) were 3.9 (3.5-4.2) and 618 (504-857) in LTNPs, 3.3 (2.8-4.7) and 463 (333-627) in SPs, and 4.6 (4.3-5.3) and 201 (110-254) in RPs. Gp41 and C2-V3 amino acid divergence was lower in LTNP and SP strains compared to RPs (median value: 0.085 and 0.091 vs. 0.114, p = 0.005 and 0.042) and a trend of lower variability was observed for Nef (p = 0.198). A lower entropy value was observed at 10, 3, and 7 positions of Gp41, C2-V3, and Nef belonging to LTNPs and at 7, 3, and 1 positions of Gp41, C2-V3, and Nef belonging to SPs compared with RPs (p < 0.05). Focusing on epitope rich regions, again a higher degree of conservation was observed in Gp41 and C2-V3 sequences belonging to LTNPs and SPs compared to those belonging to RPs. This study shows that the extent of amino acid variability correlates with a different HIV-1 progression rate. This variability also involves CTL epitope rich regions, thus suggesting its involvement in the immune escape process modulation.

First external quality assurance program of the Italian HLA-B*57:01 Network assessing the performance of clinical virology laboratories in HLA-B*57:01 testing.

BACKGROUND: Since the HLA-B*57:01 allele is strongly associated with abacavir hypersensitivity reaction, testing for the presence of HLA-B*57:01 is mandatory before administration of abacavir. While HLA-B*57:01 testing is usually provided by pharmacogenetics, genetics or blood transfusion services, clinical virology laboratories can be an optimal opportunity for HLA-B*57:01 testing since they receive blood samples for routine HIV monitoring and have the expertise for convenient and less expensive PCR-based point mutation assays. OBJECTIVES: The Italian HLA-B*57:01 Network gathers accredited clinical virology laboratories offering HLA-B*57:01 testing in Italy with the aim to share protocols, test new methods, develop and maintain external quality assurance (EQA) programs. STUDY DESIGN: A panel of 9HLA-B*57:01-positive and 16HLA-B*57:01-negative frozen blood samples were blindly distributed to 10 units including 9 clinical virology laboratories and one reference pharmacology laboratory. Each laboratory was free to use its own routine method for DNA extraction and HLA-B*57:01 testing. RESULTS: DNA was extracted by automated workstations in 6 units and by manual spin columns in 4. Eight units used the Duplicα Real Time HLA-B*57:01 kit by Euroclone and two units used two different PCR homemade protocols. All the 10 units correctly identified all the 25 samples. CONCLUSIONS: The first HLA-B*57:01 EQA program run in Italy showed that clinical virology units are equipped and proficient for providing HLA-B*57:01 testing by inexpensive assays easy to integrate into their routine.

Potential implications of CYP3A4, CYP3A5 and MDR-1 genetic variants on the efficacy of Lopinavir/Ritonavir (LPV/r) monotherapy in HIV-1 patients.

INTRODUCTION: Several genetic single nucleotide polymorphisms (SNPs) in biotransformation enzymes (CYP3A4, CYP3A5) or transporter proteins (multidrug resistance MDR1 gene product, P-gp) are involved in PI metabolism so that PI pharmacokinetics is characterized by a large inter-individual variability. The aim of this study was: (i) to develop an in-house PCR/direct sequencing, based on DNA purification of full-length CYP3A4 and CYP3A5 genes (SNPs) and MDR1 C3435T variant; (ii) to investigate association of CYP3A4 and CYP3A5 reported or unreported genetic polymorphisms and MDR1-C3435T (CC homozygote, CT heterozygote, TT homozygote) with clinical outcome of HIV-1 infected subjects treated with PI. METHODS: Overall, 39 HIV-1 infected patients receiving boosted Lopinavir (LPV/r) monotherapy after virological suppression were genotyped and analyzed through PCR and direct sequencing of full-length CYP3A4 and CYP3A5 gene sequences (1) and MDR1 gene (C3435T). CD4+T-cell counts and plasma viral load were analyzed before and after LPV/r initiation; LPV/r therapeutic drug monitoring (TDM) was determined at 12-hours. RESULTS: LPV/r TDM (ng/ml) did not show significant differences among CYP3A4 or CYP3A5 SNPs, although a mean lower level of LPV/r was associated with detection of several SNPs: CYP3A5*3 rs776746; CYP3A5 rs28365088, CYP3A5 rs15524, CYP3A4 rs2687116, and a not already described polymorphism CYP3A4 nt20338. In follow-up analysis, <90% adherence was the main factor associated with virological failure of LPV/r monotherapy (83.3% of failure vs 34.4%, p<0.001 at log-rank test). Adjusting for adherence, the detection of a single CYP3A5*3 rs776746 and CYP3A5 rs15524 SNPs was associated with higher probability of LPV/r monotherapy failure (p<0.01), and in general, detection of any CYP3A5 SNP was associated with failure (26.2% vs 58.3%, p=0.067). No-association with detection of any CYP3A4 SNPs was found. MDR1 TT variants showed significant lower frequency of treatment failure (0.0% vs 47.7%, p=0.026), since non-TT homozygote patient failed LPV/r monotherapy. CONCLUSIONS: Efficacy of PI monotherapy is strongly dependent from patient adherence, but, in adherent patients, genetic factors, such as CYP3A5 and MDR1-C3435T gene variants, may affect the response to treatment, though their role, as well of other genetic variants, need further investigation.

Analysis of single-nucleotide polymorphisms (SNPs) in human CYP3A4 and CYP3A5 genes: potential implications for the metabolism of HIV drugs.

BACKGROUND: Drug metabolism via the cytochrome P450 (CYP450) system has emerged as an important determinant in the occurrence of several drug interactions (adverse drug reactions, reduced pharmacological effect, drug toxicities). In particular, CYP3A4 and CYP3A5 (interacting with more than 60% of licensed drugs) exhibit the most individual variations of gene expression, mostly caused by single nucleotide polymorphisms (SNPs) within the regulatory region of the CYP3A4 and CYP3A5 genes which might affect the level of enzyme production.In this study, we sought to improve the performance of sensitive screening for CYP3A polymorphism detection in twenty HIV-1 infected patients undergoing lopinavir/ritonavir (LPV/r) monotherapy. METHODS: The study was performed by an effective, easy and inexpensive home-made Polymerase Chain Reaction Direct Sequencing approach for analyzing CYP3A4 and CYP3A5 genes which can detect both reported and unreported genetic variants potentially associated with altered or decreased functions of CYP3A4 and CYP3A5 proteins. Proportions and tests of association were used. RESULTS: Among the genetic variants considered, CYP3A4*1B (expression of altered function) was only found in 3 patients (15%) and CYP3A5*3 (expression of splicing defect) in 3 other patients (15%). CYP3A5*3 did not appear to be associated with decreased efficacy of LPV/r in any patient, since none of the patients carrying this variant showed virological rebound during LPV/r treatment or low levels of TDM. In contrast, low-level virological rebound was observed in one patient and a low TDM level was found in another; both were carrying CYP3A4*1B. CONCLUSIONS: Our method exhibited an overall efficiency of 100% (DNA amplification and sequencing in our group of patients). This may contribute to producing innovative results for better understanding the inter-genotypic variability in gene coding for CYP3A, and investigating SNPs as biological markers of individual response to drugs requiring metabolism via the cytochrome P450 system.

Reliability and clinical relevance of the HIV-1 drug resistance test in patients with low viremia levels.

BACKGROUND: We evaluated reliability and clinical usefulness of genotypic resistance testing (GRT) in patients for whom combination antiretroviral therapy (cART) was unsuccessful with viremia levels 50-1000 copies/mL, for whom GRT is generally not recommended by current guidelines. METHODS: The genotyping success rate was evaluated in 12 828 human immunodeficiency virus type 1 (HIV-1) plasma samples with viremia >50 copies/mL, tested using the commercial ViroSeq HIV-1 Genotyping System or a homemade system. Phylogenetic analysis was performed to test the reliability and reproducibility of the GRT at low-level viremia (LLV). Drug resistance was evaluated in 3895 samples from 2200 patients for whom treatment was unsuccessful (viremia >50 copies/mL) by considering the resistance mutations paneled in the 2013 International Antiviral Society list. RESULTS: Overall, the success rate of amplification/sequencing was 96.4%. Viremia levels of 50-200 and 201-500 copies/mL afforded success rates of 67.2% and 88.1%, respectively, reaching 93.2% at 501-1000 copies/mL and ≥97.3% above 1000 copies/mL. A high homology among sequences belonging to the same subject for 96.4% of patients analyzed was found. The overall resistance prevalence was 74%. Drug resistance was commonly found also at LLV. In particular, by stratifying for different viremia ranges, detection of resistance was as follows: 50-200 copies/mL = 52.8%; 201-500 = 70%; 501-1000 = 74%; 1001-10 000 = 86.1%; 10 001-100 000 = 76.7%; and >100 000 = 63% (P < .001). Similar bell-shaped results were found when the GRT analysis was restricted to 2008-2012, although at a slightly lower prevalence. CONCLUSIONS: In patients failing cART with LLV, HIV-1 genotyping provides reliable and reproducible results that are informative about emerging drug resistance.

Performance of genotypic tropism testing on proviral DNA in clinical practice: results from the DIVA study group.

OBJECTIVE: The DIVA study is aimed at setting up a standardized genotypic tropism-testing on proviral-DNA for the routine clinical diagnostic-laboratory. METHODS: Twelve local centres and 5 reference centres (previously cross-validated) were identified. For inter-center validation-procedure, 60 peripheral-blood mononuclear cells (PBMCs) aliquots from 45 HAART-treated patients were randomly chosen for population V3 sequencing on proviral-DNA at local HIV centre and at reference-laboratory. Viral tropism was predicted by Geno2Pheno algorithm (False Positive Rate [FPR] = 20%) as proposed by the European-Guidelines. Quantification of total HIV-1 DNA was based on a method described by Viard (2004). RESULTS: Quantification of HIV-1 DNA was available for 35/45 (77.8%) samples, and gave a median value of 598 (IQR:252- 1,203) copies/10 PBMCs. A total of 56/60 (93.3%) samples were successfully amplified by both the reference and the local virological centers. The overall concordance of tropism prediction between local and reference centers was 54/56 (96.4%). Results of tropism prediction by local centers were: 33/54 (61.1%) R5 and 21/54 (38.9%) X4/DM. CONCLUSION: There was high concordance in the genotypic tropism prediction based on proviral DNA among different virological centers throughout Italy. Our results are in line with other European studies, and support the use of genotypic tropism testing on proviral DNA in patients with suppressed plasma HIV-1 RNA candidate to CCR5-antagonist treatment.

Comparative analysis of drug resistance among B and the most prevalent non-B HIV type 1 subtypes (C, F, and CRF02_AG) in Italy.

In recent years, increasing numbers of patients infected with HIV-1 non-B subtypes have been treated with modern antiretroviral regimens. Therefore, a better knowledge of HIV drug resistance in non-B strains is crucial. Thus, we compared the mutational pathways involved in drug resistance among the most common non-B subtypes in Italy (F, C, and CRF02_AG) and the B subtype. In total, 2234 pol sequences from 1231 virologically failing patients from Central Italy were analyzed. The prevalence of resistance mutations in protease and reverse transcriptase between non-B and B subtypes has been evaluated. Among patients treated with nucleoside/nucleotide reverse transcriptase inhibitors (NRTI) and with thymidine analogues (TA) experience, TAMs1 M41L and L210W were less prevalent in CRF02_AG, while TAMs2 T215F and K219E were more prevalent in the F subtype. In NRTI-treated patients having experience with abacavir, didanosine, tenofovir, or stavudine the K65R mutation was mostly prevalent in the C subtype. In non-NRTI (NNRTI)-treated patients infected by the C subtype the prevalence of K103N was lower than in patients infected with other subtypes, while the prevalence of Y181C and Y188L was higher compared to subtype B. The prevalence of Y181C was higher also in subtype F as compared to subtype B. In patients treated with protease inhibitors, L89V was predominantly found in CRF02_AG, while the TPV resistance mutation T74P was predominantly found in the C subtype. Some differences in the genotypic drug resistance have been found among patients infected with B, C, F, and CRF02_AG subtypes in relationship to treatment. These results may be useful for the therapeutic management of individuals infected with HIV-1 non-B strains.

Study of genotypic and phenotypic HIV-1 dynamics of integrase mutations during raltegravir treatment: a refined analysis by ultra-deep 454 pyrosequencing.

BACKGROUND: The dynamics of raltegravir-resistant variants and their impact on virologic response in 23 HIV-1-infected patients, who started a salvage raltegravir-containing regimen, were investigated. METHODS: Integrase population sequencing and Ultra-Deep-454 Pyrosequencing (UDPS) were performed on plasma samples at baseline and at raltegravir failure. All integrase mutations detected at a frequency ≥1% were considered to be reliable for the UDPS analyses. Phylogenetic and phenotypic resistance analyses were also performed. RESULTS: At baseline, primary resistance mutations were not detected by both population and UDPS genotypic assays; few secondary mutations (T97A-V151I-G163R) were rarely detected and did not show any statistically association either with virologic response at 24-weeks or with the development of resistant variants at failure. At UDPS, not all resistant variants appearing early during treatment evolved as major populations during failure; only specific resistance pathways (Y143R-Q148H/R-N155H) associated with an increased rate of fitness and phenotypic resistance were selected. CONCLUSIONS: Resistance to raltegravir in integrase strand transfer inhibitor-naive patients remains today a rare event, which might be changed by future extensive use of such drugs. In our study, pathways of resistance at failure were not predicted by baseline mutations, suggesting that evolution plus stochastic selection plays a major role in the appearance of integrase-resistance mutations, whereas fitness and resistance are dominant factors acting for the late selection of resistant quasispecies.

Identification and structural characterization of novel genetic elements in the HIV-1 V3 loop regulating coreceptor usage.

BACKGROUND: The interaction between HIV-1 gp120 and CCR5 N terminus is critical for R5-virus entry and affects CCR5 antagonists' activity. Knowledge of how different genetic signatures of gp120 V3 domain effect the strength of this interaction is limited. METHODS: HIV-1 coreceptor usage was assessed in 251 patients using enhanced-sensitivity Trofile assay and V3 sequencing plus tropism prediction by Geno2pheno algorithm. Bayesian partitional model and recursive model selection have been used to define V3 genetic determinants correlated with different coreceptor usage. Gp120 interaction with CCR5 N terminus was evaluated by docking-analysis/molecular-dynamic simulations starting from the model described previously. RESULTS: Selected V3 genetic determinants (beyond known aminoacidic positions) significantly correlate with CCR5- or CXCR4-usage, and modulate gp120 affinity for CCR5 N terminus. This is the case for N5Y and N7K, absent in CCR5-using viruses and present in 4.5% and 6% of CXCR4-using viruses, respectively, and A19V, occurring in 2.6% of CCR5-using viruses and 22.0% of CXCR4-using viruses (P=10(-2) to 10(-7)). Their presence determines a decreased affinity for CCR5 N terminus even stronger than that observed in the presence of the well-known mutation S11R (N5Y: -6.60 Kcal/mol; N7K: -5.40 Kcal/mol; A19V: -5.60 Kcal/mol; S11R: -6.70 Kcal/mol; WT: -6.90 Kcal/mol). N7K significantly increases the distance between V3 position 7 and sulphotyrosine at CCR5 position 14 (crucial for binding to gp120; from 4.22 Å to 8.30 Å), thus abrogating the interaction between these two important residues. CONCLUSIONS: Key determinants for tropism within the V3 sequence, confirmed by structure- and by phenotypic-tropism, have been identified. This information can be used for a finer tuning of potential efficacy of CCR5-antagonists in clinical practice, and to provide molecular implications for design of new entry inhibitors.

Selected amino acid mutations in HIV-1 B subtype gp41 are associated with specific gp120v3 signatures in the regulation of co-receptor usage.

BACKGROUND: The third variable loop (V3) of the HIV-1 gp120 surface protein is a major determinant of cellular co-receptor binding. However, HIV-1 can also modulate its tropism through other regions in gp120, such as V1, V2 and C4 regions, as well as in the gp41 protein. Moreover, specific changes in gp41 are likely to be responsible for of damage in gp120-CCR5 interactions, resulting in potential resistance to CCR5 inhibitors.In order to genetically characterize the two envelope viral proteins in terms of co-receptor usage, we have analyzed 526 full-length env sequences derived from HIV-1 subtype-B infected individuals, from our and public (Los Alamos) databases. The co-receptor usage was predicted by the analysis of V3 sequences using Geno2Pheno (G2P) algorithm. The binomial correlation phi coefficient was used to assess covariation among gp120V3 and gp41 mutations; subsequently the average linkage hierarchical agglomerative clustering was performed. RESULTS: According to G2P false positive rate (FPR) values, among 526 env-sequences analyzed, we further characterized 196 sequences: 105 with FPR <5% and 91 with FPR >70%, for X4-using and R5-using viruses, respectively.Beyond the classical signatures at 11/25 V3 positions (S11S and E25D, R5-tropic viruses; S11KR and E25KRQ, X4-tropic viruses), other specific V3 and gp41 mutations were found statistically associated with the co-receptor usage. Almost all of these specific gp41 positions are exposed on the surface of the glycoprotein. By the covariation analysis, we found several statistically significant associations between V3 and gp41 mutations, especially in the context of CXCR4 viruses. The topology of the dendrogram showed the existence of a cluster associated with R5-usage involving E25DV3, S11SV3, T22AV3, S129DQgp41 and A96Ngp41 signatures (bootstrap = 0.88). Conversely, a large cluster was found associated with X4-usage involving T8IV3, S11KRV3, F20IVYV3, G24EKRV3, E25KRV3, Q32KRV3, A30Tgp41, A189Sgp41, N195Kgp41 and L210Pgp41 mutations (bootstrap = 0.84). CONCLUSIONS: Our results show that gp120V3 and several specific amino acid changes in gp41 are associated together with CXCR4 and/or CCR5 usage. These findings implement previous observations that determinants of tropism may reside outside the V3-loop, even in the gp41. Further studies will be needed to confirm the degree to which these gp41 mutations contribute directly to co-receptor use.

Molecular epidemiology of HIV type 1 CRF02_AG in Cameroon and African patients living in Italy.

HIV-1 CRF02_AG accounts for >50% of infected individuals in Cameroon. CRF02_AG prevalence has been increasing both in Africa and Europe, particularly in Italy because of migrations from the sub-Saharan region. This study investigated the molecular epidemiology of CRF02_AG in Cameroon by employing Bayesian phylodynamics and analyzed the relationship between HIV-1 CRF02_AG isolates circulating in Italy and those prevalent in Africa to understand the link between the two epidemics. Among 291 Cameroonian reverse transcriptase sequences analyzed, about 70% clustered within three distinct clades, two of which shared a most recent common ancestor, all related to sequences from Western Africa. The major Cameroonian clades emerged during the mid-1970s and slowly spread during the next 30 years. Little or no geographic structure was detected within these clades. One of the major driving forces of the epidemic was likely the high accessibility between locations in Southern Cameroon contributing to the mobility of the population. The remaining Cameroonian sequences and the new strains isolated from Italian patients were interspersed mainly within West and Central African sequences in the tree, indicating a continuous exchange of CRF02_AG viral strains between Cameroon and other African countries, as well as multiple independent introductions in the Italian population. The evaluation of the spread of CRF02_AG may provide significant insight about the future dynamics of the Italian and European epidemic.

HIV-1 dual/mixed tropic isolates show different genetic and phenotypic characteristics and response to maraviroc in vitro.

Dual/mixed-tropic HIV-1 strains are predominant in a significative proportion of patients, though few information is available regarding the genetic characteristics, quasispecies composition, and susceptibility against CCR5-antagonists of the primary-isolates. For this reason, we investigated in deep details, both phenotypically and genotypically, the characteristics of 54 HIV-1 primary-isolates obtained from HIV-infected patients. Tropism was assessed by multiple-cycles phenotypic-assay on U87MG-CD4(+)-CCR5(+)-/CXCR4(+)-expressing cells. In vitro selection in PBMCs of X4-tropic viral strains following maraviroc-treatment was also performed. Phenotypic-assay reported pure R5-tropic viruses in 31 (57.4%) isolates, dual/mixed-tropic viruses in 22 (40.7%), and pure X4-tropic virus in only 1 (1.8%). Among dual/mixed-tropic isolates, 12 showed a remarkably higher replication-efficacy in CCR5-expressing cells (R5(+)/X4), and 2 in CXCR4-expressing cells (R5/X4(+)). Genotypic-tropism testing showed a correlation between PSSM-scores, geno2pheno false-positive-rate, and V3-net-charge with both CCR5-usage and syncytium-inducing ability. Moreover, specific gp120- and gp41-mutations were significantly associated with tropism and/or syncytium-inducing ability. Ultra-deep V3-pyrosequencing showed the presence of a swarm of genetically distinct species with a preference for CCR5-coreceptor not only in all pure R5-isolates, but also in 6/7 R5(+)/X4-tropic isolates. In both pure-X4 and R5/X4(+)-isolates, we observed extensive prevalence of X4-using species. In vitro selection-experiments with CCR5-inhibitor maraviroc (up to 2 months) showed no-emergence of X4-tropic variants for all R5- and R5(+)/X4-isolates tested (while X4-virus remained fully-resistant). In conclusion, our study shows that dual/mixed-tropic viruses are constituted by different species, whereby those with characteristics R5(+)/X4 are genotypically and phenotypically similar to the pure-R5 isolates; thus the use of CCR5-antagonists in patients with R5(+)/X4-tropic viruses may be a therapeutic-option that deserves further investigations.

Performance of genotypic tropism testing in clinical practice using the enhanced sensitivity version of Trofile as reference assay: results from the OSCAR Study Group.

OBJECTIVE: The goal of the OSCAR programme is to evaluate the performances of genotypic HIV-1 tropism testing in clinical practice using the enhanced sensitivity version of Trofile (ESTA) as reference-assay. METHODS: HIV-1 coreceptor-usage was assessed using plasma samples from 406 HIV-1 infected patients by ESTA and by gp120 V3 population-sequencing followed by Geno2pheno (set at a False Positive Rate [FPR] of 10% and 5%). RESULTS: ESTA was successful in 365 (89.9%) samples indicating R5 in 254 (69.6%), and DM/X4 in 111 (30.4% of samples (104 [28.5%] DM and 7 [1.9%] X4). Genotypic-testing successfully assessed viral tropism for all 406 samples, including the 41 with undetermined result by ESTA. Genotypic-tropism testing at a FPR of 5% and 10% was 81.1% and 78.4% concordant with ESTA, respectively. Despite a sensitivity of 48.7% and 55.9% at a FPR of 5% and 10%, respectively, a high concordance (specificity: 95.3% for FPR of 5% and 88.2% for FPR of 10%) between genotypic-tropism testing and ESTA was reached in the detection of R5-tropic viruses. CONCLUSION: Our results are in line with other European studies, and support the routine use of genotypic tropism testing in clinical-settings for monitoring of HIV-1 infected patients candidate to or failing CCR5-antagonists.

Specific HIV-1 integrase polymorphisms change their prevalence in untreated versus antiretroviral-treated HIV-1-infected patients, all naive to integrase inhibitors.

OBJECTIVES: To define whether the prevalence of mutations associated with integrase inhibitor (INI) resistance is different in untreated versus antiretroviral-treated HIV-1-infected individuals (all INI naive). METHODS: Gene sequences of the integrase (IN) and reverse transcriptase (RT) obtained from plasma samples of a well-defined cohort of 448 HIV-1-infected individuals (134 drug naive and 314 antiretroviral treated) were analysed. Docking simulations, using RT and IN models, were also performed. RESULTS: Primary mutations and the majority of secondary mutations for raltegravir or elvitegravir were completely absent (or rarely found, <1%) in INI-naive patients, either drug naive or antiretroviral treated. Specific IN polymorphisms increased their frequency in antiretroviral-treated patients, and showed positive associations with specific RT resistance mutations. M154I and V165I IN polymorphisms occurred at a frequency of 6% in untreated patients, reaching 21.3% and 13.4%, respectively, in antiretroviral-treated patients. The mutation M154L, absent in drug-naive patients, was prevalent at 5.7% in antiretroviral-treated patients, and was positively associated with RT resistance mutations F227L and T215Y. Similarly, V165I and G163R mutations were associated with the RT resistance mutations F227L and M230L, respectively, and the T206S polymorphism was associated with the RT resistance mutation L210W. Docking simulations showed several favourable contacts between IN and RT residues. CONCLUSIONS: Overall, results confirm that primary and secondary INI-associated mutations are absent or extremely rare in INI-naive patients. Conversely, a few specific IN polymorphisms found in INI-naive patients increased their frequency in antiretroviral-failing patients and/or are associated with RT resistance mutations. The potential contribution of such polymorphisms to the evolution of resistance under the pressure of INIs needs further investigation.

Use of novel antiretroviral agents in rescue regimens: a case of early virological failure to raltegravir.

In patients with virological failure during highly active antiretroviral therapy (HAART) and drug resistance, guidelines recommend the achievement of maximal virological suppression by the use of a new regimen with at least 2 active drugs. We describe the clinical outcome of a heavily antiretroviral-experienced patient who experienced early failure to raltegravir.

Characterization of the patterns of drug-resistance mutations in newly diagnosed HIV-1 infected patients naïve to the antiretroviral drugs.

BACKGROUND: The transmission of HIV-1 drug-resistant strains in drug naive patients may seriously compromise the efficacy of a first-line antiretroviral treatment. To better define this problem, a study in a cohort of newly diagnosed HIV-1 infected individuals has been conducted. This study is aimed to assess the prevalence and the patterns of the mutations recently associated with transmitted drug resistance in the reverse transcriptase (RT) and in protease (PR) of HIV-1. METHODS: Prevalence of transmitted drug resistant strains is determined in 255 newly diagnosed HIV-1 infected patients enrolled in different counselling and testing (CT) centres in Central Italy; the Avidity Index (AI) on the first available serum sample is also used to estimate time since infection. Logistic regression models are used to determine factors associated with infection by drug resistant HIV-1 strains. RESULTS: The prevalence of HIV-1 strains with at least one major drug resistance mutation is 5.9% (15/255); moreover, 3.9% (10/255) of patients is infected with HIV nucleoside reverse transcriptase inhibitor (NRTI)-resistant viruses, 3.5% (9/255) with HIV non-NRTI-resistant viruses and 0.4% (1/255) with HIV protease inhibitor (PI)-resistant viruses. Most importantly, almost half (60.0%) of patients carries HIV-1 resistant strains with more than one major drug resistance mutation. In addition, patients who had acquired HIV through homosexual intercourses are more likely to harbour a virus with at least one primary resistance mutation (OR 7.7; 95% CI: 1.7-35.0, P = 0.008). CONCLUSION: The prevalence of drug resistant HIV-1 strains among newly diagnosed individuals in Central Italy is consistent with the data from other European countries. Nevertheless, the presence of drug-resistance HIV-1 mutations in complex patterns highlights an additional potential risk for public health and strongly supports the extension of wide genotyping to newly diagnosed HIV-1 infected patients.

Characterization and structural analysis of HIV-1 integrase conservation.

The HIV-1 integrase, responsible for the chromosomal integration of the newly synthesized double-stranded viral DNA into the host genomic DNA, represents a new and important target of potential clinical relevance. For instance, two integrase inhibitors, raltegravir and elvitegravir, have been shown to be promising in clinical trials, and the first has been recently made available for clinical practice. As is the case for other antiviral drugs, drug resistance to integrase inhibitors occurs both in vitro and/or in vivo through the selection of mutations within the HIV genome. Indeed, many integrase mutations have already been associated with resistance to all the different integrase inhibitors tested in in vitro and/or in vivo studies. Among them, about 40 substitutions have been specifically associated with the development of resistance to raltegravir and/or elvitegravir; some of them were also found in vivo in patients failing such integrase inhibitors. The relevance of integrase mutations in clinical practice has yet to be defined, in light of the lack of long-term follow-up of treated patients and the limited data about the prevalence of integrase inhibitor-associated mutations in integrase inhibitor-naive patients (either untreated, or treated with antiretrovirals not containing integrase inhibitors). Therefore, by structural analysis elaboration and literature discussion, the aim of this review is to characterize the conserved residues and regions of HIV-1 integrase and the prevalence of mutations associated with integrase inhibitor resistance, by matching data originated from a well-defined cohort of HIV-1 B subtype-infected individuals (untreated and antiretroviral-treated) and data originated from the public Los Alamos Database available in the literature (all patients integrase inhibitor-naive by definition). In integrase inhibitor-naive patients, 180 out of 288 HIV-1 integrase residues (62.5%) are conserved (< 1% variability). Residues involved in protein stability, multimerization, DNA binding, catalytic activity, and in the binding with the human cellular cofactor LEDGF/p75 are fully conserved. Some of these residues clustered into large defined regions of consecutive invariant amino acids, suggesting that consecutive residues in specific structural domains are required for the correct performance of HIV-1 integrase functions. All primary signature mutations emerging in patients failing raltegravir (Y143R, Q148H/K/R, N155H) or elvitegravir (T66I, E92Q, S147G, Q148H/K/R, N155H), as well as secondary mutations (H51Y, T66A/K, E138K, G140S/A/C, Y143C/H, K160N, R166S, E170A, S230R, D232N, R263K) were completely absent or highly infrequent (< 0.5%) in integrase inhibitor-naive patients, either infected with HIV-1 B subtype (drug-naive or antiretroviral-treated), or non-B subtypes/group N and O. Differently, other mutations (L74M, T97A, S119G/R, V151I, K156N, E157Q, G163K/R, V165I, I203M, T206S, S230N) occurred as natural polymorphisms with a different prevalence according to different HIV-1 subtype/circulating recombinant form/group. In conclusion, the HIV-1 integrase in vivo is an enzyme requiring the full preservation of almost two-thirds of its amino acids in the absence of specific integrase inhibitor pressure. Primary mutations associated with resistance to integrase inhibitors clinically relevant today are absent or highly infrequent in integrase inhibitor-naive patients. The characterization of the highly conserved residues (involved in protein stability, multimerization, DNA binding, catalytic activity, LEDGF binding, and some with still poorly understood function) could help in the rational design of new HIV-1 inhibitors with alternative mechanisms of action and more favorable resistance profiles.

Dynamics of NRTI resistance mutations during therapy interruption.

Abstract To date, very little information is available regarding the evolution of drug resistance mutations during treatment interruption (TI). Using a survival analysis approach, we investigated the dynamics of mutations associated with resistance to nucleoside analogue reverse transcriptase inhibitors (NRTIs) during TI. Analyzing 132 patients having at least two consecutive genotypes, one at last NRTI-containing regimen failure, and at least one during TI, we observed that the NRTI resistance mutations disappear at different rates during TI and are lost independently of each other in the majority of patients. The disappearance of the K65R and M184I/V mutations occurred in the majority of patients, was rapid, and was associated with the reemergence of wild-type virus, thus showing their negative impact on viral fitness. Overall, it seems that the loss of NRTI drug resistance mutations during TI is not an ordered process, and in the majority of patients occurs without specific interaction among mutations.

Treatment with the fusion inhibitor enfuvirtide influences the appearance of mutations in the human immunodeficiency virus type 1 regulatory protein rev.

The gp41-encoding sequence of the env gene contains in two separate regions the Rev-responsive elements (RRE) and the alternative open reading frame of the second exon of the regulatory protein Rev. The binding of Rev to the RRE allows the transport of unspliced/singly spliced viral mRNAs out of the nucleus, an essential step in the life cycle of human immunodeficiency virus type 1 (HIV-1). In this study, we have investigated whether the fusion-inhibitor enfuvirtide (ENF) can induce mutations in Rev and if these mutations correlate with the classical ENF resistance gp41 mutations and with viremia and CD4 cell count. Specific Rev mutations were positively associated with ENF treatment and significantly correlated with classical ENF resistance gp41 mutations. In particular, a cluster was observed for the Rev mutations E57A (E57A(rev)) and N86S(rev) with the ENF resistance gp41 mutations Q40H (Q40H(gp41)) and L45M(gp41). In addition, the presence at week 48 of the E57A(rev) correlates with a significant viremia increase from baseline to week 48 and with a CD4 cell count loss from baseline to week 48. By modeling the RRE structure, we found that the Q40(gp41) and L45(gp41) codons form complementary base pairs in a region of the RRE involved in Rev binding. The conformation of this Rev-binding site is disrupted when Q40H(gp41) and L45M(gp41) occur alone while it is restored when both mutations are present. In conclusion, our study shows that ENF pressure may also affect both Rev and RRE structures and can provide an excellent example of compensatory evolution. This highlights the multiple roles of ENF (and perhaps other entry inhibitors) in modulating the correct interplay between the different HIV-1 genes and proteins during the HIV-1 life cycle.

Human immunodeficiency virus type 1 intersubtype recombinants predominate in the AIDS epidemic in Cameroon.

A broad and rapidly changing HIV Type 1 (HIV-1) diversity has been reported from different populations in Cameroon since the early epidemic. Our understanding of HIV-1 dynamics can be improved by a systematic surveillance in Cameroon as accessibility and use of antiretroviral drugs increase. To contribute to this, we genotyped 30 samples by sequencing the protease and reverse transcriptase (proRT) genes of HIV-1. Phylogenetic analysis of the HIV-1 proRT sequences using the MEGA3 software showed that 26 (86.7%) were recombinant forms which included 20 (66.7%) circulating recombinant forms: CRF02_AG, (50%), CRF06_cpx (3.3%), CRF11 _cpx (10%) and CRF37_cpx (3.3%), and 6 unique recombinant forms (URF, 20%). Two of the six URFs were second generation recombinants and 4 contained unclassified segments. HIV-1 subtypes A1 (3.3%), C (3.3%) and D (6.7%) were also identified. Although partial sequences of HIV-1 genome were analysed, our results indicate that recombinant HIV-1 variants predominate in the AIDS epidemic in Cameroon. With the widespread use of antiretroviral drugs in Cameroon and the circulation of several HIV-1 variants within this population, the emergence of recombinants with unknown diagnostic and clinical consequences is a concern.