HIV-DNA: nuovo marcatore virologico. Metodiche a confronto per la quantificazione di HIV-DNA

Transcription

1 HIV-DNA: nuovo marcatore virologico Metodiche a confronto per la quantificazione di HIV-DNA Maria Carla Re Laboratorio Retrovirus e Agenti infettivi HIV correlati UO di Microbiologia, Università di Bologna Alma Mater Studiorum

2 HIV DNA: a new must for HIV patients In latently infected cells the proviral DNA, integrated in the host s genome, does not actively replicate becomes invisible to the host immune system is unaffected by existing antiviral drugs is responsible of viral rebound The HIV-1 reservoir is highly complex memory CD4+ T cells are the most infected cells and different T-cell subsets macrophages,microglial cells, lymphoid organs and gastrointestinal tract have been found to harbor latent infection

3 HIV DNA predictor of the outcome of HIV-1 infection Early treatments lead to low levels of HIV-1 DNA It is a potential indicator for the initiation of antiretrovirals High levels were associated to rapid progression to AIDS and death. Association was independent of HIV- 1 RNA levels and CD4 counts A low DNA load (<10 copies/ml ) is a strong marker to exclude HIV-1 associated nephropathy and HIV-1 neurocognitive disorders Riva et al., 2003; Goujard C et al. 2006; Nicastri E et al., 2008, Coiras et al 2009; Masquelier et al., 2011; Sarmati et al 2012; Lambert-Niclot et al 2012, Demetriou et 2012; Tsiara et al

4 The perfect HIV-1 DNA quantification assay offer higher sensitivity and specificity should also be cost-effective technically easy to implement in clinical laboratories Proviral HIV-1 DNA is detectable in three major forms that reflect the different stages and fates of development during viral replication: the linear nonintegrated form the circular nonintegrated form found exclusively in the nucleus as 1-LTR and 2-LTR based on the number of LTR in HIV-1 DNA. half-life: short or long the linear integrated provirus. linear DNA: also called provirus Stable enough during the infection course Nucleus Transcribed or latent state

5 Nonintegrated HIV-1 DNA linear the most abundant form It is the direct product of reverse transcribed viral RNA and is the substrate for the integration reaction. Nonintegrated HIV-1 DNA circular 1-LTR circles 2-LTR circles Besides integrated DNA or proviral DNA, various forms of unintegrated cdna exist in the infected cell The relative proportion of integrated to unintegrated forms can vary from 1:1 to 1:100 in vitro considered to be the dead end products of abortive infections Indicative of recent viral infection (2-LTR circles: on the basis of the notion that these circles are unstable) Predictor of disease progression and decreased response to antiretroviral therapy.

6 Integrated HIV-1 DNA (provirus) and non integrated DNA Which is the right choice? Total HIV-1 DNA (integrated and non integrated forms) Integrated HIV -1 DNA (provirus) Long half life Long half life Short half-life Unintegrated HIV-1 DNA It is not always a suitable estimate of proviral DNA (in treated patients) monitoring of therapeutic approaches Is significantly lower in elite suppressors than that found in HAART treated patients Is a better indicator of reservoir size. Treated patients often have excess of unintegrated forms 2-LTR circles might be a marker of nascent infections (?) 2-LTR increase circle levels during drug intensification are indicative of ongoing viral replication 2-LTR circles are exclusively found in the nucleus, they have become a useful marker of viral nuclear import in studies of viral trafficking

7 Total HIV-1 DNA Real-time PCR has been the most common for quantification of total (integrated and nonintegrated HIV-1 DNA) by in-house protocols 1. by using many targets including pol gene, gag gene or gag-pol junction and also the conserved region of the long terminal repeat (LTR) [more sensitive, specific and reproducible than pol or gag based Assays] 2. by PCR optimized with 2 sets of primers targeting LTR and gag 3. by methods based on primers that amplify the viral sequence between the 5 LTR region and the gag gene The sensitivity of these different in-house techniques is expressed in various ways, making comparisons difficult between studies. The measure of total HIV-1 DNA should then approach the level of integrated HIV-1 DNA in patients on HAART since unintegrated HIV-1 DNA has a short half-life

8 Total HIV-1 DNA: a couple of examples Correlations of pre-art HIV-DNA with outcome in first-line treated ART patients Total HIV-DNA: modified version of a commercial Kit (Surdo et al 2015) Results, normalized by CD4 +, show that: Pre-ART HIV-DNA content in CD4+ cells strongly correlated with baseline viroimmunologic parameters and inflammation markers. Baseline HIV-DNA was also found to predict virological rebound in a cohort of 1st-line ART. 3 real-time PCR protocols (pol real-time PCR, gag real-time PCR, LTR real-time PCR ) LTR real-time PCR: higher correlation between PBMCs HIV-1 DNA and viremia The presence of mismatches between primers, probes and patients sequences is the most frequent and reasonable reason for under-evaluation or lack of detection (primers and probes are located in genomic regions non-conserved or involved in drug resistance) Ceccherini-Silberstein et al., ICONA at CROI 2016 Rozera et al. JVM 2010

9 Author Sample Target Detection method Control gene Estimated LOQ Desiré 2001 PBMCs pol Taqman probe Albumine 10 cp/10 6 PMBC Ericksson 2003 CD4 pol Taqman probe Albumine 2 cp/10 4 CD4+ Gibellini 2004 PBMCs pol SYBR green Globin 5 cp/ PBMCs Zhao 2002 PBMCs gag Taqman probe Internal QS 10 cp/10 6 PBMCs Lillo 2004 PBMCs commercial Taqman probe DNA QS DNA-QS 1.7 log 10 cp/106 PBMCs Gibellini 2008 PBMCs gag SYBR green Globin 5cp/PCR reaction Kabamba 2005 CD4 Gag /pol Taqman probe Globin 5cp/PCR reaction Pasternak 2008 PBMCs gag Taqman probe Beta-actin 4 cp/pcr reaction Avettand Fenoel 2009 PBMCs /W.B LTR Molecular beacons none 40 cp/10 6 PBMCs Beloukas 2009 PBMCs LTR /gag Molecular beacons CCR5 gene 12.5 copies/10 6 PBMCs Kostrikis 2002 PBMCs From 5 LTR to gag Taqman probe CCR5 gene 10 copies/106 6 PBMCs Casabianca 2007 W.blood From 5 LTR to gag SYBR green none 2 cp/mg of DNA Modified from E.K. Alidjinou et al., 2015

10 Integrated HIV-1 DNA Several PCR assays such as inverse PCR, linker ligation PCR (not strictly quantitative) More sensitive alternative (Alu-gag PCR) combined a pre-amplification step (performed with primers that bind genomic Alu elements and HIV-1 gag sequences) and a nested real-time PCR that quantities HIV-1 LTR sequences using molecular beacon detection The sensitivity of this method was increased by incorporating a repetitive sampling technique to detect rare integration events that occur near an Alu repeat.

11 HIV nonintegrated DNA HIV-1 LTR Amplification of 1-LTR circles is complicated by the inherent homology of the LTR sequence present in all cdna forms Primers designed for 1-LTR circle detection were able to amplify spurious products from other cdna forms including linear cdna molecules and integrated provirus HIV nonintegrated DNA HIV-2 LTR The 2-LTR circles have been readily quantified by PCR assays, due to its unique LTR- LTR junction Stable or not? It has been argued that 2-LTR circles are quickly degraded in HIV -infected cells However the presence of 2-LTR circles to be used as a marker for ongoing de novo infection in patients. By the use of fluorescence-monitored PCR (Taqman) to quantitate the metabolism of HIV cdna early after infection. Contrary to previous work, it was found that 2-LTR circles are actually quite stable (Butler et al.2002)

12 HIV nonintegrated DNA HIV-2 LTR Total HIV-1 DNA, unspliced (us) and multiply spliced HIV-1 RNA, and 2LTR circles can be quantified by digital PCR in peripheral blood Digital PCR is an alternative to real-time PCR in which each sample is divided into thousands of independent microscopic reactions prior to PCR amplification Kiselinova et al., PLoS Pathog. 2016

13 An unsolved question: HIV detected by PCR is a competent or a defective virus? Pt PBMC Limiting dilution culture PHA activation and g irradiated PBMC 1 2 Resting CD4 cells PBMC are collected from HIV-1 infected individuals and resting CD4 + T cells (CD25, CD69, HLA-DR ) are purified. Resting T cells are plated in 5-fold serial dilutions in duplicate from 1,000,000 to 320 cells per well. To reverse latency HIV-1 provirus, patient cells are activated with PHA and a 10-fold excess of irradiated PBMC from healthy donors Laird et al. Plos Pathog 2013 Target cells for HIV-1 infection are added to allow outgrowth of replication-competent HIV-1 released from infected cells in which latency has been reversed. In the MOLT-4/CCR5 viral outgrowth assay, MOLT-4/CCR5 cells are added on day 2 only. Day 2 Day 7 standard assay, HIV-1 p24 antigen ELISA is used to identify wells positive for HIV-1 outgrowth at 14 days. For the MOLT-4/CCR5 assay, RT-PCR is used to identify wells positive for outgrowth at 7 days.

14 Another possibility: the Tilda assay (Tat/rev Induced Limiting Dilution Assay) A novel assay to measure the magnitude of the inducible viral reservoir in HIV-infected individuals. To measure the frequency of cells with inducible multiply-spliced HIV RNA, as these transcripts are usually absent in latently infected cells but induced upon viral reactivation. TILDA requires less than a million cells, does not require RNA extraction and can be completed in two days. PBMCs isolation CD4 + T cells isolation. CD4 + T cells counted and plated (a 96 well plate) or stimulated for 12 h with PMA/ionomycin. Tat/rev msrna were quantified by an ultrasensitive nested RT-PCR and the frequency of cells with inducible msrna was determined Procopio et al Biomedicine 2015

15 Is it possible to predict HIV-1 eradication in latent reservoir? A 2,000-fold reductions are required to permit a majority of patients to interrupt ART for 1 y without rebound and that rebound may occur suddenly after multiple years. Greater than 10,000-fold reductions may be required to prevent rebound altogether. Alison L. Hilla et al. PNAS 2014 The stochastic viral decision. Each viral infection is a roll of the dice, with some probability of entering latency whatever the cellular activation state. Weinberger and Weinberger, Cell 2013

16 Four future statements To create a national network to obtain a well standardized method for HIV DNA detection, with high sensitivity and reproducibility (establishing a cut-off Total, integrated and nonintegrated DNA) To introduce, in the next guidelines, the suggestion (or the priority) for a quantitative test for HIV DNA detection (and to establish which forms) before therapy and during patients follow-up To study the presence of competent and/or defective HIV in reservoir Don t forget HIV DNA test in babies from HIV infected mothers Thanks to I. Bon, G. Musumeci, A. Bertoldi, S. Longo, G. Gallinella and D. Gibellini