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Andreis, Samantha (2014) Studio longitudinale dell'evoluzione del tropismo per X4/R5, dei livelli di HIV-1 DNA cellulare, dei parametri immunologici e della viremia residua in una popolazione di pazienti naive trattati con successo. [Tesi di dottorato]

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Abstract (inglese)

Background:
Nowadays HIV-1 RNA levels and CD4+ T lymphocyte counts are the standard markers used in clinical practice for the management of HIV infection. However the evolution of HIV infection can be monitored also by measuring HIV-DNA and this measurement can be determined in PBMCs, even during powerful and prolonged antiretroviral therapy.
In successfull treated HIV-1 patients, viral load is undetectable and the strategies for managing long-term side effects may involve a new class of antiretroviral-like CCR5 antagonists. Moreover the dynamics and the influence of viral tropism on the course of HIV-1 infection in subjects exposed to antiretroviral therapy are not fully understood. Then the evolution and determination of HIV-1 tropism based on cellular DNA sequence could be useful for patients with a successfully suppressed plasma viral load.

Aims: In this study we aimed to determine whether the HIV-1 tropism for CXCR4 or CCR5 correlates with residual viraemia, cellular HIV-1 DNA load and CD4+ count; moreover, we evaluated if exist a correlation between baseline and follow-up HIV-1 DNA levels with residual viraemia, baseline plasma HIV-1 RNA, and the condition of primary or chronic HIV infection at the start of antiretroviral therapy.

Methods: In the CAVeAT, that is a prospective cohort of HIV-infected patients enrolled starting from 2004 in five infectious diseases units in Northeastern Italy (Veneto region), we retrospectively selected two subgroup of patients (cohort I and cohort II); they were a subset of subjects achieving virological suppression within 6 months after initiation of first-line therapy and maintaining plasma HIV RNA levels < 50 copies/ml, without virological failures, until evaluation at the follow-up time points. In order to be included in the our study, the patients needed to be naïve and treated with effective antiretroviral therapy. None of the patients were treated with CCR5 antagonists. The cohort I consisted on 219 patients with median follow-up time of 3 years (T0, T1, T2) while the cohort II was represented of 181 patients with median follow-up times of 4 years (T0, T1, T2, T3). Genotypic analysis of viral tropism was performed on PBMCs throught the sequencing of V3 loop of gp120; the generated sequences were interpreted using the bioinformatic tool Geno2pheno coreceptor while proviral DNA was quantified by Real-Time PCR using TaqMan probes.

Results: In the cohort I, HIV-1 DNA, CD4+ count and plasma viraemia were available from all 219 patients at T0 and T1, and in 86 subjects at T2, while tropism determinations were available from 109 subjects at T0, 219 at T1, and from 86 subjects at T2. The results showed that achieving a residual viraemia < 2.5 cp/ml at T1 correlated with having the same condition at T2 and that there was a positive correlation between To and T1 -T2 tropism. X4 tropism at T1 negatively correlated with the possibility of achieving viraemia < 2.5 cp/ml at T2 while a positive correlation between viremic suppression and R5 coreceptor affinity was found.
In 181 patients of the cohort II, viroimmunological data were collected at baseline (T0) and at two follow-up time points (T1, T2); in a subgroup of 70 subjects, we evaluated also a third follow-up time point (T3). We observed that high baseline plasma HIV-1 RNA values positive correlated with high levels of HIV-1 DNA at T0, T1, T2, T3 and negative correlated with residual viraemia at T1, T2, T3; having high levels of HIV-1 DNA at T0 positive correlated with high values at T1, T2, T3 and negatively correlated with achieving residual viraemia. Primary infection was associated with lower probability of having high HIV-1 DNA levels at T1, T2, T3 and with a higher probability of achieving residual viraemia at T1 and T3, with respect to chronic infection.

Conclusions: The tropism of archived virus was stable during an effective treatment, although a low percentage of patients switched over time. R5 tropism and its stability were related to achieving and maintaining viraemia < 2.5 copies/ml, in treatment responder patients, suggesting a relation among viral tropism and response to treatment in the long term.
Moreover, we demonstrated a strong and long-lasting correlation between viral load and cellular HIV-1 DNA before and after the start of HAART and that cellular HIV-1 DNA is closely related to residual viraemia over long-term follow-up of ART responders, particularly when treated during primary infection.

Abstract (italiano)

Background: Fino ad oggi nella pratica clinica la gestione dell'infezione da HIV-1 ha utilizzato come marcatori prognostici i livelli di RNA plasmatico e il numero dei linfociti CD4+. Tuttavia nei pazienti trattati, l'evoluzione dell'infezione da HIV può essere valutata anche misurando l'HIV-1 DNA nei reservoirs.
In pazienti HIV-1 trattati con terapia di successo, la carica virale è non rilevabile e le strategie per la gestione degli effetti collaterali a lungo termine potrebbero considerare l'utilizzo di una nuova classe di antiretrovirali antagonisti del CCR5. Inoltre non è ancora stato del tutto chiarito come il tropismo virale e la sua evoluzione influenzino il decorso dell'infezione da HIV-1 in soggetti in terapia antiretrovirale. Quindi per valutare come gestire i pazienti trattati con successo nei quali la carica virale è soppressa, potrebbe essere utile determinare l'evoluzione del tropismo di HIV-1 sul DNA cellulare.

Scopo: In questo studio di coorte costituita da pazienti naive trattati con successo abbiamo valutato se il tropismo per CXCR4 o CCR5 di HIV-1 correla con la viremia residua; inoltre abbiamo determinato se esiste una correlazione tra i livelli di HIV-1 DNA cellulare (al baseline e ai follow-up) con la viremia residua, l'HIV-1 RNA al baseline, e con il fatto di avere un'infezione acuta all'inizio della terapia antiretrovirale.

Materiali e metodi: All'interno della coorte CAVeAT, che è una coorte prospettica di pazienti HIV positivi arruolati a partire dal 2004 da cinque Unità di Malattie Infettive della regione Veneto, sono stati retrospettivamente selezionati due sottogruppi di pazienti (coorte I e coorte II); sono tutti pazienti che hanno raggiunto una soppressione virologica entro i 6 mesi dopo il primo trattamento e hanno mantenuto livelli di HIV-1 RNA al di sotto di 50 copie/ml senza fallimento virologico per tutti i tempi di follow-up. Per essere eleggibili, i pazienti dovevano essere naive e poi essere trattati con una terapia antiretrovirale di successo. Nessuno dei pazienti doveva essere in trattamento con gli antagonisti del CCR5. La coorte I era costituita da 219 pazienti con una mediana di follow-up di 3 anni (T0, T1, T2) mentre la coorte II era rappresentata da 181 pazienti con una media di follow up di 4 anni (T0, T1, T2, T3). Le analisi genotipiche per il tropismo virale sono state eseguite su PBMCs mediante sequenziamento del loop V3 di gp120; le sequenze ottenute sono state interpretate utilizzando l'algoritmo bioinformatico Geno2pheno coreceptor mentre il DNA provirale è stato quantificato mediante Real-Time PCR utilizzando sonde TaqMan.

Risultati: Nella coorte I, la valutazione dell'HIV-1 DNA, della conta dei CD4+ e della viremia è stata effettuata su tutti i 219 pazienti al T0 e T1, e su 86 soggetti al T2; il tropismo è stato determinato solo in 109 soggetti al T0, su tutti i 219 al T1, e in 86 pazienti al T2. I risultati hanno mostrato che il raggiungere una viremia residua al di sotto di 2.5 cp/ml al T1 correlava con il suo mantenimento al T2 e che c'era una correlazione positiva tra il tropismo al To e quello ai follow-up ( T1-T2 ). Avere un tropismo X4 al T1 correla negativamente con la possibilità di raggiungere una viremia residua al di sotto di 2.5 cp/ml al T2 mentre è stata trovata una correlazione positiva tra soppressione virologica e tropismo R5.
Nei 181 pazienti della coorte II, i dati viro-immunologici sono stati eseguiti per tutti i soggetti al baseline (T0) e ai due tempi di follow-up (T1, T2); in un sottogruppo di 70 pazienti, abbiamo preso in considerazione anche un terzo tempo di follow-up (T3). Quindi abbiamo osservato che alti livelli di HIV-1 RNA al baseline correlavano positivamente con alti livelli di HIV-1 DNA al T0, T1, T2, T3 e negativamente con la viremia residua al T1, T2, T3; avere alti livelli di HIV-1 DNA al T0 correlava positivamente con alti livelli anche al T1, T2, T3 e negativamente con il raggiungimento della viremia residua al di sotto delle 2.5 copie/ml. Avere un'infezione acuta, piuttosto che cronica, all'inizio della terapia era associato con una più bassa probabilità di avere alti livelli di HIV-1 DNA al T1, T2, T3 e con una più alta probabilità di raggiungere una viremia residua al T1 e T3.

Conclusioni: Il tropismo del virus archiviato è rimasto stabile durante tutto il periodo della terapia, sebbene in una bassa percentuale di pazienti sia cambiato nel tempo. In pazienti trattati con una terapia di successo, il tropismo R5 e la sua stabilità sono stati confrontati con il fatto di raggiungere e mantenere una viremia residua al di sotto di 2.5 copie/ml, suggerendo una relazione tra il tropismo virale e la risposta ad una terapia a lungo termine. Inoltre, è stata dimostrata una forte correlazione tra la carica virale e l'HIV-1 DNA prima e dopo l'inizio della terapia antiretrovirale (ART) e che l'HIV-1 DNA cellulare è strettamente connesso alla viremia residua
nel follow-up a lungo termine nei soggetti rispondenti alla terapia, in modo particolare se trattati durante l'infezione acuta, ovvero il prima possibile.

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Tipo di EPrint:Tesi di dottorato
Relatore:Parisi, Saverio Giuseppe
Dottorato (corsi e scuole):Ciclo 26 > Scuole 26 > BIOMEDICINA
Data di deposito della tesi:01 Luglio 2014
Anno di Pubblicazione:01 Luglio 2014
Parole chiave (italiano / inglese):HIV/HIV, tropismo/tropism, HIV DNA/HIV DNA load, viremia residua/residual viraemia, clinical study/studio clinico, cohort study/studio corte
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/07 Microbiologia e microbiologia clinica
Struttura di riferimento:Dipartimenti > Dipartimento di Medicina Molecolare
Codice ID:6937
Depositato il:04 Ago 2015 11:56
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