Emanuela Giombini

Detection of quasispecies variants predicted to use CXCR4 by ultra-deep pyrosequencing during early HIV infection.

Objectives:HIV-1 V3 quasispecies was analyzed by ultra-deep pyrosequencing, in early HIV-infected patients, to assess possible correlations between quasispecies diversity, frequency of variants predicted to use CXCR4 and need for early antiretroviral treatment. Methods:Twenty patients were retrospectively enrolled: 10 patients (group A) required HAART within 6 months from seroconversion and 10 (group B) remained free of therapy during this period. V3 quasispecies was assessed on plasma viral RNA and in peripheral blood mononuclear cell-associated proviral DNA. Prediction of coreceptor usage was performed by position-specific score matrix analysis. Results:Variants predicted to use CXCR4 were detected (frequency ≥0.3%) in the plasma of 50% of early infected patients (60% from group A and 40% from group B). Intrapatient frequency of these variants was highly variable (0.3–56.3%). A positive correlation was observed between the proportion of X4 variants and intrapatient quasispecies diversity. Quasispecies diversity and absolute numbers of X4 variants were significantly higher in patients from group A. The analysis of proviral DNA quasispecies, performed in a subgroup of five patients, showed that X4 variants were not detected in patients with RNA frequency below 0.3%, and detected at 3.6% in the patient with 56.3% of X4 plasma variants. Conclusion:Our findings show that X4 variants may be frequently found, at variable intrapatient frequency, in early infected patients, and that quasispecies diversity and absolute numbers of X4 variants are significantly higher in patients undergoing early antiretroviral treatment. Further studies are mandatory to explore the clinical relevance of X4 variants present during early infection with respect to clinical progression and possible therapeutic implications.

Role of HIV-1 matrix protein p17 variants in lymphoma pathogenesis.

Significance Non-Hodgkin lymphomas (NHLs) are associated with HIV-1 infection, but the HIV1 genome is not detectable in malignant B cells. Here we show that variants of the HIV-1 matrix protein p17 (vp17s) are detected in the NHL specimens of HIV+ patients. These vp17s are more frequently detected in HIV+ patients with NHL than in patients without NHL. These vp17s display a potent B-cell growth-promoting activity, which is exerted by activating the Akt signaling pathway. Results obtained by CD spectroscopy and thermal denaturation suggest that mutation-induced protein destabilization may lead to a conformational change potentially responsible for the viral protein to promote B-cell growth. Our results suggest that vp17s may have a role in sustaining lymphomagenesis, thus offering new opportunities to prevent and/or treat HIV-associated NHLs. Although in decline after successful anti-HIV therapy, B-cell lymphomas are still elevated in HIV-1-seropositive (HIV+) persons, and the mechanisms are obscure. The HIV-1 matrix protein p17 persists in germinal centers long after HIV-1 drug suppression, and some p17 variants (vp17s) activate Akt signaling and promote growth of transformed B cells. Here we show that vp17s derived from four of five non-Hodgkin lymphoma (NHL) tissues from HIV+ subjects display potent B-cell growth-promoting activity. They are characterized by amino acid insertions at position 117–118 (Ala–Ala) or 125–126 (Gly–Asn or Gly–Gln–Ala–Asn–Gln–Asn) among some other mutations throughout the sequence. Identical dominant vp17s are found in both tumor and plasma. Three of seven plasma samples from an independent set of NHL cases manifested multiple Ala insertions at position 117–118, and one with the Ala–Ala profile also promoted B-cell growth and activated Akt signaling. Ultradeep pyrosequencing showed that vp17s with C-terminal insertions are more frequently detected in plasma of HIV+ subjects with than without NHL. Insertion of Ala–Ala at position 117–118 into reference p17 (refp17) was sufficient to confer B-cell growth-promoting activity. In contrast, refp17 bearing the Gly–Asn insertion at position 125–126 did not, suggesting that mutations not restricted to the C terminus can also account for this activity. Biophysical analysis revealed that the Ala–Ala insertion mutant is destabilized compared with refp17, whereas the Gly–Asn form is stabilized. This finding provides an avenue for further exploration of structure function relationships and new treatment strategies in combating HIV-1–related NHL.

Extent of HCV NS3 protease variability and resistance-associated mutations assessed by next generation sequencing in HCV monoinfected and HIV/HCV coinfected patients

HCV quasispecies variability represents the background for the selection of mutations and for the development of drug resistance. Natural aminoacid changes in NS3, associated with reduced protease inhibitor susceptibility, have been observed in treatment-naïve patients. Massively parallel sequencing has been used to analyze NS3 quasispecies in patients infected with HCV genotype 1, naive to anti-HCV treatment, with/without HIV-coinfection, to establish the genetic heterogeneity and the presence of amino acid substitutions at positions responsible for drug resistance. Genomes carrying substitutions represented either predominant or minority components of viral quasispecies, and were observed in 85.7% of patients. Multiple substitutions, frequently associated on the same haplotype, were observed in 46.4% of patients. High resistance combinations were not detected, neither on the same genome, nor in the whole quasispecies. Heterogeneity of HCV NS3 was lower in HIV-coinfected as compared to HCV-monoinfected patients, but factors underlying this difference remain to be established. Although the relevance of naturally occurring mutations with respect of resistance development and probability of success of direct acting antivirals is questioned, UDPS may be beneficial to help understanding viral dynamics, providing high resolution view of viral diversity.

Ultra-deep sequencing reveals hidden HIV-1 minority lineages and shifts of viral population between the main cellular reservoirs of the infection after therapy interruption

Viral quasispecies population dynamics between monocytes and T‐lymphocytes were analyzed in patients after highly active antiretroviral therapy (HAART) interruption, during a follow‐up of 3–6 months. V3 env region underwent ultra‐deep pyrosequencing. Co‐receptor usage prediction was performed by Position Specific Score Matrix Analysis. Phylogenetic trees were constructed to evaluate the relationships between the variants. Gene flow was also investigated. Even though at the moment of therapy interruption monocyte‐derived HIV‐1 genomes presented higher genetic heterogeneity than that of T‐lymphocytes, at subsequent times, this difference in genetic heterogeneity disappeared, due to different waves of expansion and reduction of quasispecies variability associated with monocytes and T‐lymphocytes. Phylogenetic analysis and gene flow evaluation supported the hypothesis of extensive interchange of variants between cellular compartments of the infection. A spread of proviral X4 lineages hidden in monocytes to T cells was observed, but this was not associated with an overall shift towards CXCR4 using variants during the observation period. J. Med. Virol. 84:839–844, 2012.

Dynamics of HCV genotype 4 resistance-associated variants during virologic escape with pIFN/RBV + daclatasvir: A case study using ultra deep pyrosequencing

BACKGROUND Daclatasvir (DCV) is an approved NS5A inhibitor with potent anti-HCV activity and broad genotype coverage. DCV resistance-associated variants (RAVs) have been described for patients infected with genotype (GT) 1, but increased GT4 prevalence in European countries as a result of immigration has boosted interest in this genotype. OBJECTIVES Establishment of NS5A variability in treatment-naive patients with HCV genotype 4 infection and a case study of the dynamics of resistance-associated variants in a virologic failure receiving pIFN/RBV+DCV, as assessed by ultra-deep sequencing. STUDY DESIGN Five treatment-naïve GT4 patients (GT4a [n = 1], GT4d [n = 3], GT4o [n = 1]) were evaluated for inclusion in the COMMAND-4 study and treatment with pIFN/RBV±DCV. RESULTS Patient (Pt) 1 received pIFN/RBV; Pts2-4 received pIFN/RBV + DCV; Pt5 was a screening failure. Pt1 relapsed; Pt2 experienced breakthrough at Wk4; Pts3 and 4 achieved a sustained virologic response. No substitutions associated with DCV-resistance were detected at baseline. In terms of viremic time points for Pts1 and 2, the extent of NS5A diversity pre-treatment was not significantly related to viral load (r = -0568; p = 0.035). In Pt2, multiple substitutions associated with DCV-resistance were observed after breakthrough at NS5A amino acid positions 28, 31 and 93. These substitutions were frequently observed on the same haplotype (L28S + M31I = 55.52, 82.50, and 99.36% at Wk4, 8 and 9; L28S + M31I + Y93H = 11.77, 5.01 and <0.6% at Wk4, 8 and 9). CONCLUSIONS This is the first report to describe DCV-resistance in patients infected with GT4d, supporting a possible role for a recently described RAV (L28S), and presenting the dynamics of HCV quasispecies during therapy failure, with indications of changes of diversity and association of mutations.

Structural dynamics of V3 loop with different electrostatics: implications on co-receptor recognition: a molecular dynamics study of HIV gp120

The HIV’s envelope glycoprotein gp120 plays a major role in the entry of the virus into the host cell, through its successive interactions with the cell surface CD4 receptor and a co-receptor (CCR5 or CXCR4). The choice of a specific co-receptor by gp120 has an important consequence on HIV infection and pathogenesis. The third variable region within gp120, the V3 loop, is the principal determinant of the co-receptor usage by gp120. Here, we report the long time molecular dynamics simulations of four gp120 structures, having a V3 loop charge of +3 and +5, from both R5 and X4 specific strains of HIV. The results of the study highlight the properties of the V3 loop that can be critical for dictating the co-receptor recognition and selection in structural context. In detail, we observe that the structural orientation of the V3 loop in the 3D space is modulated by its net charge, whilst its co-receptor choice is likely dictated by a combined effect of both the electrostatics of the loop and its conformational variability at the level of its central crown region.

Quasispecies tropism and compartmentalization in gut and peripheral blood during early and chronic phases of HIV-1 infection: possible correlation with immune activation markers

HIV quasispecies was analysed in plasma and proviral genomes hosted by duodenal mucosa and peripheral blood cells (PBMC) from patients with early or chronic infection, with respect to viral heterogeneity, tropism compartmentalization and extent of immune activation. Seventeen HIV-1-infected combined antiretroviral therapy naive patients were enrolled (11 early infection and six chronic infection). V3 and nef genomic regions were analysed by ultra-deep pyrosequencing. Sequences were used to infer co-receptor usage and to construct phylogenetic trees. As markers of immune activation, plasma sCD14 and soluble tumour necrosis factor receptor II (sTNFRII) levels were measured. Median diversity of HIV RNA was lower in patients with early infection versus chronic infection patients. Overall, direct correlation was observed between V3 diversity and X4 frequency; V3 diversity of HIV RNA was inversely correlated with CD4 T-cell count; median sCD14 and sTNFRII values were similar in early and chronic patients, but X4 frequency of HIV RNA was directly correlated with plasma sCD14. The proportion of patients harbouring X4 variants and median intra-patient X4 frequency of proviral genomes tended to be higher in chronic infection than early infection patients. More pronounced compartmentalization of proviral quasispecies in gut compared with PBMC samples was observed in patients with early infection compared with chronic patients. The loss of gut/PBMC compartmentalization in more advanced stages of HIV infection was confirmed by longitudinal observation. More studies are needed to understand the pathogenetic significance of early HIV quasispecies compartmentalization and progressive intermixing of viral variants in subsequent phases of the infection, as well as the role of immune activation in tropism switch.

Slow response to entecavir treatment in treatment-naive HBV patients is conditioned by immune response rather than by the presence or selection of refractory variants

BACKGROUND Entecavir is the drug of choice as first-line treatment for treatment-naive HBV patients. As a result of the high genetic barrier to resistance, treatment failure remains rare, but occurs within 3 years of initiation, suggesting that viral genetic characteristics may provide a fast lane to resistance. One of the main concerns is the long time to viral suppression observed in some (even treatment-naive) patients. The reasons for this phenomenon were investigated in a group of chronic hepatitis B treatment-naive patients. METHODS Out of 23 treatment-naive patients starting entecavir, the 5 with the best and those with the worst viral load decay curves were selected for the study. Quasispecies analysis was performed for the reverse transcriptase/hepatitis B surface antigen (HBsAg) open reading frame (ORF) by ultra-deep pyrosequencing. For each patient, the analysis was performed at baseline (T0) and when viraemia reached between 15,000 and 200 IU/ml (T1). RESULTS The few resistance mutations present at T0 were not selected by treatment; no other resistance mutations or suggestive mutational patterns were selected at T1. Selective pressure analysis indicated that both at T0 and T1 the HBsAg ORF was subjected to a significantly higher pressure in rapid responders, especially in a region rich in cytotoxic T-lymphocyte (CTL) epitopes. CONCLUSIONS The results did not provide evidence that a slower response to entecavir is due to the emergence of less sensitive variants. Rather, the lower selective pressure and variability in humoral and CTL epitopes in slow responders suggests that their immune response might be at odds in rapidly clearing infected cells from the liver.

HCV NS3 quasispecies in liver and plasma and dynamics of telaprevir-resistant variants in breakthrough patients assessed by UDPS: A case study

BACKGROUND The impact of pre-existing variants in hepatitis C virus (HCV) quasispecies, carrying resistance-associated mutations (RAMs), on the outcome of treatment with direct acting antiviral agents (DAA) is debated and it is complicated by the lack of knowledge of quasispecies distribution between the viral reservoir (liver) and the circulating compartment. OBJECTIVE To evaluate NS3 protease heterogeneity and presence of RAMs on baseline plasma and liver biopsy samples. Plasma dynamics were also analyzed during therapy and after its suspension. Study design Ultra-deep pyrosequencing (UDPS) was performed in two HCV genotype 1a patients who received telaprevir (TVR)-based therapy and developed treatment failure due to TVR-resistance. RESULTS In both patients the baseline diversity of NS3 quasispecies in plasma was higher than in liver (183.6×10(-4) vs 47.8×10(-4) and 246.0×10(-4) vs 55.0×10(-4) nt substitution/site, respectively, p<0.0001), but phylogenetic trees did not evidence compartmentalization between the two compartments. At baseline RAMs (i.e. V36A, T54A) were detected very low levels (range: 0.31-0.52%) in both specimen types. However, phylogenetic analyses revealed that the viral variants carrying these mutations at baseline were different from those that became fixed at breakthrough, when combined V36M+R155K, conferring high-level resistance to TVR, were observed. The frequency of resistance-associated variants declined after withdrawal of drug selective pressure. CONCLUSIONS UDPS allowed extensive evaluation of quasispecies compartmentalization and of their dynamics after withdrawal of TVR. Plasma and liver NS3 quasispecies, including low level RAMs, do not show significant difference.

Near full length hepatitis C virus genome reconstruction by next generation sequencing based on genotype-independent amplification

BACKGROUND Deep sequencing has a deep impact on the study of rapidly mutating RNA viruses, such as hepatitis C virus, proving to be an invaluable tool for analyzing virus diversity and evolution. AIM Genotype-independent high-throughput pyrosequencing was used to obtain near full length hepatitis C virus genome sequence reconstruction directly from clinical samples. METHODS Samples from hepatitis C virus infected subjects harbouring different subtypes (1a, 1b, 2c) were analyzed (viral load range: 1.2-20.8 × 10(6)IU/ml). Data were generated with a modified sequence-independent single primer amplification method followed by 454 sequencing. RESULTS the extent of reconstructed hepatitis C virus genome varied from 79.95% to 99.64%. No correlation between extent of genome reconstruction and either viral load (r=0.4857, p=0.3556) or number of HCV reads (r=0.08571, p=0.9194) was observed. CONCLUSION This study describes a protocol for obtaining whole genome sequences from different hepatitis C virus patients with different genotypes in a single sequencing run.