BUONGIORNO A TUTTI!!!!!!!!!! AREVIR-GenaFor-Meeting Verbundprojekt - - PowerPoint PPT Presentation
BUONGIORNO A TUTTI!!!!!!!!!! AREVIR-GenaFor-Meeting Verbundprojekt HIV-HEP-MASTER EuResist (8-9/5/2015) Comparison of HIV-1 genotypically predicted tropism between circulating plasma RNA and proviral DNA in drug-experienced patients Lavinia
Lavinia Fabeni
National Institute for Infectious Diseases L. Spallanzani - IRCCS, Rome, Italy University of Rome Tor Vergata, Italy AREVIR-GenaFor-Meeting Verbundprojekt HIV-HEP-MASTER EuResist (8-9/5/2015)
already experiencing virologic failure because of resistance to other antiretroviral agents (Rodger D. et al., CID, 2008; Sayana S. et al., Expert Rev Anti Infect Ther, 2009).
HIV-1 RNA. But in limited settings (as very low or undetectable viral load or in case
alternative source of viral genetic material for tropism testing (Vandekerckhove L. et al., Lancet Infect Dis, 2011), and would facilitate the use of CCR5 inhibitors as part of switching, simplification, or intensification strategies.
tropism prediction based on plasma RNA and pvDNA (Saracino A. et al., Virus Res, 2007; Seclén E. et al., JAC, 2010; Verhofstede C. et al., HIV Med, 2011; Paar C. et al., J Clin Microbiol, 2011; Gupta S. et al., JAC, 2013; Bon I. et al., J Med Virol, 2014), but the factors that could be associated with the discrepancy between the two compartments has not yet been clearly determined.
Paired PBMCs DNA and plasma RNA samples were obtained from 55 drug experienced subjects and were tested for the V3 region of gp120 (during 2010-2014) RNA extraction from plasma by QIAmp Viral RNA mini Kit DNA extraction from PBMCs by QIAmp DNA mini Kit Amplification by home-made RT-PCR Amplification by home-made PCR Amplification by NESTED-PCR Sequencing by Genetic Analyzer 3130
Viral RNA and proviral DNA sequences were compared at the amino acid level
period of 24 months maximum, according to the observation that no evolution is found in samples within 24 months of follow-up, both for the mutated positions and the total number of mutations (Palmisano L. et al., HIV Med, 2009).
purposes: i) determine viral subtype; ii) check any cross-contamination; iii) evaluate the proper clustering of the pvDNA and plasma RNA sequences from the same subject. The robustness of the paired samples clusters was tested using the maximum likelihood (ML) method. This was inferred with the GTR nucleotide substitution model with gamma-distribution of among site rate heterogeneity, a proportion of invariable sites (GTR+I+Г5) (Tavaré S., Lect Math. Life Sci, 1986), and 1000 bootstrap replicates. Paired samples clusters were identified by a bootstrap support >70%. Genetic distances analyses were conducted using the Tajima-Nei model (Tajima F. and Nei M., Mol Biol Evol, 1984).
(http://coreceptor.bioinf.mpi-inf.mpg.de/).
usage, genetic distance and V3 mutations prevalence.
copies/mL).
37/55 couples of samples were contextually available, while 18/55 had paired samples with blood sampling dates within a median [interquartile period, IQR] period of 7 [2-9] months.
Patient’s characteristics at the time of pvDNA V3 genotyping
3 C, 3 BF, 2 F1, 2 G, 1 BC and 1 CRF06_cpx
By phylogenetic analysis, sequences belonging to the same subject showed a high homology in 100% of cases, denoting genetic relatedness.
Maximum likelihood (ML) tree of the 55 RNA/DNA paired samples (bootstrap value >70%)
Plasma RNA pvDNA
The concordance between GTT in plasma RNA and pvDNA compartments was found in 46/55 (84%) samples (A). The concordance was very high among contextual samples (92.0%, 34/37) (B), and still considerable among non contextual samples (66.7%, 12/18) (C). Discordant samples were characterized by a higher FPR difference between pvDNA and RNA than concordant samples (median [IQR] ΔFPR (%): 20 [11-52] vs. 1 [0-7.6], p<0.001) and a higher genetic variability (median [IQR] genetic distance: 0.07 [0.02-0.23] vs. 0.00 [0.00-0.02] p=0.001). In general, lower viremia levels were found in discordant samples compared to concordant samples (median [IQR] log10 copies/mL: 1.73 [1.60-2.02] vs. 2.26 [1.92-4.12], p=0.036).
Characterization of HIV-1 tropism, viremia and genetic diversity between RNA/DNA paired samples.
83.6 92 66.7 16.4 8 33.3
10 20 30 40 50 60 70 80 90 100
Overall Contextual Non contextual
% Samples
Concordant Discordant
N (%)
46 (83.6) 9 (16.4) 34 (92.0) 3 (8.0) 12 (66.6) 6 (33.4) 2.26 (1.92-4.12) 1.73 (1.60-2.02) 2.85 (1.93-4.23) 1.73 (1.66-3.01) 2.13 (1.45-2.31) 1.62 (0.40-1.92) 1.0 (0.0-7.6) 20.0 (11.0-52.0) 1.1 (0-7) 20.2 (15.8-29.6) 0.8 (0.0-38) 21.0 (8.6-72.0) 0.00 (0.00-0.02) 0.07 (0.02-0.23) 0.00 (0.00-0.01) 0.07 (0.05-0.14) 0.02 (0.00-0.03) 0.07 (0.01-0.26) 0.02 (0.01-0.07)
Plasma HIV RNA, median (IQR) log10 copies/mL ΔFPR (%), median (IQR) Genetic distance, median (IQR)
0.01 (0.00-0.02) 0 (0-0.02)
A B C
p<0.001 p=0.001 p=0.036
Regarding the:
and pvDNA was found: Median [IQR]: 7 [5-9] and 6 [5-8], respectively; p=0.82- by Mann-Whitney test
Median [IQR]: 0 [0-4] in RNA sequences; 4 [0-8] in DNA sequences; p=0.008 by Mann-Whitney test The archived viruses may not always correspond to the most prevalent virus in plasma
Comparison of the overall G2P FPRs in both plasma RNA and pvDNA compartments showed good correlation (r=0.72, p<0.001) (A). A very good correlation was found for the 37 contextual samples (r=0.93, p<0.001) (B). Among the 18 non contextual samples the correlation was low (r=0.37 p=0.046) (C), explained by a high difference in FPR (%) between the 2 compartments (Median [IQR]:7.3 [0.2-43.5]).
Scatterplot for the comparison between RNA (x axis) and DNA (y axis) V3-based coreceptor tropism prediction.
R5 tropism was predicted in 38/46 (82.6%) couples of samples, with an FPR ranging from 13.0% to 96.1% and X4/DM in 8/46 (17.4%), with an FPR ranging from 0.2% to 8.6%. The proportion of X4/DM tropic virus in discordant samples was significantly higher in pvDNA than in plasma RNA (8/9 [88.9%] vs. 1/9 [11%], P=0.0034).
Overall tropism of concordant samples (A); X4/DM tropism of discordant samples (B).
The prevalence of discordant samples increased with the decrease of viremia levels
Evaluation of tropism concordance between RNA/DNA paired samples according to different viremia levels among the 42 paired samples with the same viremia level. p=0.2, by Squared-test for trend - any statistical significance was shown, probably due to the low number of samples analysed per each group
The prevalence of X4/DM samples increased with the decrease of viremia levels
Evaluation of X4/DM tropism among the 36 couples of samples with concordant tropism and the same viremia level. p=0.052 by Squared-test for trend.
No difference between the two compartments in the mutation prevalence was found in R5 concordant samples.
Prevalence of V3 mutations in R5 concordant DNA/RNA samples (N=38). All mutations found at the 35 V3 positions with an overall prevalence ≥ 10% were evaluated.
In X4/DM concordant samples, the V3 mutations (S11G, H13R, E25R, Q32K, H34Y) were found with a higher prevalence in DNA samples, while the V3 mutations (N5D, K10R, H13S, T22A, G24R, E25K, I26V) were mostly prevalent in plasma RNA than pvDNA, suggesting a different viral evolution in the two compartments.
Prevalence of V3 mutations in X4/DM concordant DNA/RNA samples (N=8). All mutations found at the 35 V3 positions with an overall prevalence ≥ 10% were evaluated.
GTT provides highly concordant tropism prediction between pvDNA and plasma RNA, confirming that prediction of viral tropism using PBMCs DNA is accurate and feasible. Among discordant samples, the presence of X4/DM tropic viruses in pvDNA in the setting of low viremia levels suggests that the determination of genotypic tropism in pvDNA provides useful information for the optimization of the therapy in patients with low or suppressed viremia.
Further studies are needed to determine whether the use of MVC, in those patients suppressed and with discordant tropism in the two compartments (R5 in plasma RNA and X4/dual-mixed in pvDNA), could be effective over time.
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C.F. Perno
..... The Patients University of Rome Tor Vergata, Rome Italy Policlinic of Rome Tor Vergata, Rome Italy INMI L Spallanzani, Rome, Italy
M.R. Capobianchi G:Berno