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Raltegravir Intensification, Viral Replication, HIV Reservoirs & HAART
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CROI: Back to Basics: the science at CROI focuses the tough questions that remain in HIV pathogenesis, Part 2 (focal points for new therapies.) - written by David Margolis MD, University of North Carolina - (03/03/09)
CROI: Back to Basics: the science at CROI focuses on the tough questions that remain in HIV pathogenesis - written by David Margolis, MD, University of North Carolina - (02/24/09)
Is replication completely stopped by ART?:....evidence from a detailed analysis of the residual viremia, suggests that it is archival and non-evolving. No evidence of new drug-resistance mutations has been found, despite continued viremia in the presence of ART....a preliminary finding presented a bit later at this very meeting (abstr. 423a, discussed below) immediately challenged this assertion by providing evidence of very low-level, ongoing viral replication.
Transient Increase in Episomal Viral cDNA following Raltegravir Intensification of a Stable HAART Regimen
M Buzon1, J Llibre2, J Gatell3, P Domingo4, R Paredes1,2, S Palmer5, M Sharkey6, M Stevenson6, B Clotet1,2, and Javier Martinez-Picado*1,7
1Fndn irsiCaixa, Badalona, Spain; 2Fndn Lluitra contra la SIDA, Badalona, Spain; 3Hosp Clinic IDIBAPS, Barcelona, Spain; 4Hosp Sant Pau, Barcelona, Spain; 5Swedish Inst for Infectious Disease Control, Solna; 6Univ of Massachusetts Med Sch, Worcester, US; and 7ICREA, Barcelona, Spain
Background: HAART significantly reduces plasma HIV-1 viremia to undetectable levels in most patients. However, residual viral replication or stable latent reservoirs are an obstacle to therapeutic eradication of HIV-1. The availability of new ARV classes provides an opportunity to intensify antiretroviral suppression. However, it is not known whether such intensification can perturb the viral reservoirs that persist in the face of HAART. We evaluated whether raltegravir (RAL) intensification of a 3-drug HAART regimen affected the levels of episomal, integrated, and total proviral DNA in patients with undetectable viremia.
Methods: We randomized 65 patients with <50 HIV-1 RNA copies/mL for at least 1 year either to intensify their HAART with RAL (n=44), or to continue their HAART (n = 21) for 48 weeks. HAART regimens included 2 NRTI and either a PI or a NNRTI. Viral DNA was extracted from peripheral blood mononuclear cells (PBMC) at weeks 0, 2, 4, and 12. Integrated proviral DNA was measured by real time PCR using LTR-Alu primers. Episomal cDNA forms (2LTR circles) were measured with primers that span the 2-LTR circle junction and total viral DNA forms were assayed using internal LTR primers.
Results: All subjects remained aviremic (<50 copies/mL) and had stable CD4 counts during the study period. Intriguingly, there was a transient and significant increase (p = 0.0391) in episomal HIV-1 DNA at week 2 in patients intensified with RAL compared to baseline and this was not associated with HAART composition. In contrast, total and proviral DNA levels remained stable during intensification.
Conclusions: Our results indicate that de novo viral infection continues in the face of HAART. Upon RAL intensification, nascent viral genomes are prevented from integrating and are subsequently converted to episomes. The lack of any fluctuation in total and proviral DNA upon intensification reinforces the notion that the majority of viral DNA in patients is archival and non-dynamic. At present, it is not possible to determine whether the virus that initiated the infection events in these individuals originated from a chronically infected cell or was a product of complete rounds of viral replication. Intensification with new ARV classes such as RAL offers an opportunity to further perturb the reservoir that persists in the face of HAART and to explore the goal of viral eradication._
No Decrease in Residual Viremia during Raltegravir Intensification in Patients on Standard ART
Joseph Jones*1, D McMahon1, A Wiegand2, M Kearney2, S Palmer2, S McNulty1, J Metcalf3, J Coffin2, J Mellors1, and F Maldarelli2
1Univ of Pittsburgh, PA, US; 2HIV Drug Resistance Prgm, NCI, Frederick, MD, US; and 3NIAID, NIH, Bethesda, MD, US
Background: Combination antiretroviral therapy can suppress plasma HIV-1 RNA to <50 copies/mL, but low-level viremia detected with more sensitive assays persists for prolonged periods. Residual viremia may be produced by long-lived, chronically infected cells or by ongoing, complete cycles of virus replication. To differentiate between these sources, we conducted a trial of treatment intensification with raltegravir in patients with plasma HIV-1 RNA <50 copies/mL but residual viremia 1 copies/mL.
Methods: Patients (n = 5) on combination antiretroviral therapy with NNRTI- or PI- based regimens including 2 NRTI and with stable HIV-1 RNA (<50 copies/mL plasma for >1 year) were screened for residual viremia using a sensitive HIV-1 RNA assay (single copy assay [SCA]). We enrolled 5 patients with persistent viremia (>1 copy HIV RNA/mL) in a 30-day drug intensification study with raltegravir 400 mg twice daily. Plasma for HIV-1 RNA assay was obtained weekly before, during, and after the 30-day intensification period..
Results: Enrolled patients (5 males) were diagnosed with HIV for a mean of 12..7 years (range 4 to 17.5 years) and had received combination ART for a mean of 8.9 years (range 3 to 15 years). The level of residual viremia before intensification (median, 1.9 copies/mL plasma) was similar to that found in prior studies of patients on standard combination therapy. Drug intensification was well tolerated with no reported adverse events. Overall, the median plasma HIV-1 RNA level during raltegravir intensification (3.2 copies/mL) was not different from the median pre-intensification level (p = 0.72). Further, no significant decreases in HIV-1 RNA levels were observed during drug intensification in individual patient analyses.
Conclusions: Raltegravir intensification for 30 days did not decrease the level of residual viremia in patients on stable ART. These results are inconsistent with the hypothesis that persistent viremia results from ongoing, complete cycles of viral replication. New therapeutic approaches will be required to eliminate HIV-1 reservoirs.
Evidence of Persistent Low-level Viremia in Long-term HAART-suppressed Individuals
H Hatano1, E Delwart1,2, P Norris1,2, T-H Lee2, J Dunn-Williams2, Peter Hunt*1, R Hoh1, J Martin1, M Busch1,2, and S Deeks1
1Univ of California, San Francisco, US and 2Blood Systems Res Inst, San Francisco, CA, US
Background: HAART can effectively reduce plasma HIV RNA levels to below the level of detection using conventional assays in most HIV-infected patients (<50 to 75 copies/mL). The degree to which residual low-level viremia persists during long-term HAART remains unclear, particularly as most studies using highly sensitive assays have had limited follow-up times.
Methods: The isothermal transcription-mediated amplification (TMA) (Aptima, Gen-Probe) assay was used to measure longitudinal plasma HIV RNA levels in 180 HAART-suppressed subjects from the SCOPE cohort. The sensitivity of the TMA assay is <3 RNA copies/mL when 4 replicates are performed. All TMA assays with a signal:cutoff (S/Co) ratio _1 were considered positive. In addition, a gdetunedh or less-sensitive enzyme immunoassay (LS-EIA) was used to obtain quantitative HIV antibody levels (measured as standardized optical density [SOD]) over time..
Results: The median pre-HAART CD4+ T cell nadir count was 100 cells/mm3. Median duration of HIV infection was 12 years. Median duration of suppressive HAART was 13 months (IQR 3.9 to 46 months); 36 subjects were suppressed for _5 years. A total of 1606 TMA assays were performed on 438 specimens (median 3 replicates per specimen) from 180 HAART-suppressed subjects. A mixed effect linear model showed an average of 0.06 decrease in S/Co per month (p <0.001); however, there was no significant change in plasma viremia over time when the analysis was limited to subjects who had been suppressed for _12 months (n = 92, average of 0.03 decrease in S/Co per month, p = 0.097) or _60 months (n = 36, average of 0.10 decrease in S/Co per month, p = 0.445). The proportion of subjects with low-level detectable viremia after 1 year of viral load suppression was 76% to 87% and did not appear to change over 7 years of suppression. Similarly, a mixed effect linear model showed an average of 0.03 decrease in HIV antibody levels per month (p = 0.006); however, there was no significant change over time when the analysis was limited to subjects who had been suppressed for _12 months (n = 34)..
Conclusions: Residual viremia is commonly observed in long-term HAART-suppressed patients, and appears to remain stable after 1 year of viral load suppression. HIV antibody levels are also stable during long-term HAART, suggesting a steady-state relationship between virus and the host response.
No Evidence for Decay in the Latent Reservoir in HIV-infected Patients Receiving Intensive Enfuvirtide-containing ART
Rajesh Gandhi*1, R Bosch2, E Aga2, M Albrecht3, E Adams4, L Demeter5, B Bastow6, R Siliciano7, J Siliciano7, J Eron8, and ACTG A5173
1Massachusetts Gen Hosp, Boston, US; 2Harvard Sch of Publ Hlth, Boston, MA, US; 3Beth Israel Deaconess Hosp, Boston, MA, US; 4NIH, Bethesda, MD, US; 5Univ of Rochester, NY, US; 6Social & Sci Systems Inc, Silver Spring, MD, US; 7Johns Hopkins Hosp, Baltimore, MD, US; and 8Univ of North Carolina at Chapel Hill, US
Background: HIV persists in resting memory CD4 T cells in patients receiving ART, with an estimated half-life of 44 months. The stability of this latent reservoir may be due to ongoing HIV replication. We assessed whether initiation of intensive ART with a multi-target regimen that includes fusion, protease and reverse transcriptase inhibitors leads to decay in the HIV latent reservoir.
Methods: In a single-arm, exploratory study, treatment-naive HIV-infected patients with a CD4 cell count 100/mm3 and viral load (VL) 1000 copies/mL initiated therapy with enfuvirtide (ENF)/tenofovir (TDF)/emtricitibine (FTC)/saquinivir (SQV)/ritonavir (RTV). Subjects who achieved a VL<50 continued ART and were tested every 24 weeks for the frequency of latently-infected resting, memory CD4 T cells for a planned duration of 96 weeks. We analyzed the latent reservoir decay rate in subjects who attained a VL <50 and remained on ENF-containing ART for at least 48 weeks.
Results: Nineteen subjects initiated intensive ART. The median baseline CD4 cell count was 262/mm3 and the median baseline VL was 4.8 log10 (63,000) copies/mL. Only 1 subject discontinued study treatment because of toxicity (pain related to injections). Seventeen subjects (89%) achieved a VL<50 within 48 weeks. Nine subjects had both virologic suppression and continued ENF-containing ART for at least 48 weeks; these subjects contributed a median of 4 latent reservoir measurements each. The 9 subjects did not differ from subjects who discontinued ENF prior to week 48 (n = 7) or did not have virologic suppression (n = 3) in terms of age, gender, baseline CD4 cell count or VL. Of the suppressed, ENF-treated subjects, four had a slight decay in the number of latently-infected cells and five subjects had a slight increase. Overall, there was no evidence for decay in the latent reservoir (95% confidence interval for half-life: 11 months to infinity).
Conclusions: In previously treatment-naive HIV-infected patients who received ENF-containing ART, we did not detect a decline in latent reservoir size over 96 weeks. We can exclude, with 95% certainty, that the half-life of latent reservoir decay with intensified, suppressive therapy is <11 months. This stability of the latent reservoir, which is similar to that seen in previous studies of less intensive ART, suggests that new strategies are needed to eradicate HIV from the latent reservoir.
Stability of HIV Reservoir Resting CD4 T Cell Subsets under Effective HAART: The rectal HIV DNA level is similar to that found in the blood intermediate subset of TEM, which are the major cells found in rectal T4, suggesting a major role of this subset in constituting HIV reservoir.
Veronique Avettand-Fenoel*1, B Descours2, L Hocqueloux3, T Prazuck3, A Samri2, A Melard1, C Blanc4, F Oualid2, B Autran2, and C Rouzioux1
1Universite Paris 5, EA3620, AP-HP, Paris, France; 2Universite Paris 6, AP-HP, Paris, France; 3Ctr Hosp Univ Orleans, France; and 4Flow Cytometry Platform, Inter IFR-UPMC, Paris, France
Background: HIV reservoir is a major obstacle to eradication. A better knowledge of which cells constitute this latent reservoir and how they evolved during time is thus necessary. We aimed at studying the resting CD4 T cells constituting the reservoir under HAART with performant technology separating different cell populations and ultra-sensitive HIV DNA quantification.
Methods: One patient with hemochromatosis, infected with HIV for 2 years, with 3.3log copies/million peripheral blood mononuclear cells (PBMC) during the primary infection symptoms and receiving effective HAART for this time was included. HIV RNA was always <20 copies/mL under treatment. Blood was then collected 4 times during 5 months, with stable HIV DNA (1.7log copies/million PBMC) and >600 CD4 T cells/μL. Resting CD4 T cells were sorted according to anti-CD45RA, CD27, and CCR7 combinations in various memory subsets between central (TCM) to effector memory (TEM), naive, and effector T cells. Ultrasensitive HIV DNA quantification was performed on the different subsets. A rectal biopsy was also collected the same day as the fourth bleed and after sorting, HIV DNA was quantified in CD4 T cells. Mann-Whitney test was used to compare results.
Results: A high stability was observed in HIV DNA levels in the different subsets over time. Virus was almost equally contained in the CD4 TEM subsets: in median values, TEM hosts 2.77log copies/million TEM [2.70 to 2.92], and the intermediate subset of TEM, 2.67log [2.57 to 2.92]. Those populations are more infected than TCM (2.18 [1.74 to 2.32]), effector (2.49 [2.06 to 2.52]), naive (1.43log [1.34 to 1.49]) (p <0.05). Among CD4 resting cells in these blood samples, taking into account the representativity of each cell subset, an intermediate subset of TEM contributes significantly more to HIV reservoir than the other (median 60% and p <0.05 in regard to all the subsets). Interestingly, HIV DNA level was 2.76log copies/million rectal CD4 T cells. Among these cells, 75% were the same intermediate subset of TEM.
Conclusions: The repartition of blood HIV reservoir is stable among the different cell subsets in a patient on effective HAART. This blood reservoir is not focused on CD4 TCM but on an intermediate subset of TEM, characterized by its activation, differentiation, and functional status. The rectal HIV DNA level is similar to that found in the blood intermediate subset of TEM, which are the major cells found in rectal T4, suggesting a major role of this subset in constituting HIV reservoir.
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