• Viral load blips can result in resistance
• IL-2 does not appear to clear HIV from lymoh node
• After viral load rebound, virus from various reservoirs may become activated

T. W. CHUN*^{1}, R. DAVEY^{1}, M. OSTROWSKI^{1}, D. ENGEL^{1}, J. MULLINS^{2}, C. LANE^{1}, and A. FAUCI^{1}. ^{1}NIAID, NIH, Bethesda, MD and ^{2}Univ. of Washington, Seattle.

The persistence of a pool of latently infected, resting CD4^{+} T cells carrying replication-competent HIV has been well documented in HIV-infected individuals who are receiving highly active antiretroviral therapy (HAART) and in whom successful suppression of plasma viremia has been achieved. Although this latent viral reservoir is considered to be a major impediment to complete eradication of HIV in infected individuals receiving HAART, the pathologic significance of this viral reservoir as well as its role in the rebound of plasma viremia after discontinuation of HAART is largely unknown. Using quantitative coculture and heteroduplex tracking assays, the authors data indicate that the pool of latently infected, resting CD4^{+} T cells does not account entirely for the early rebounding plasma HIV in infected individuals in whom HAART has been discontinued, even after prolonged periods of successful suppression of plasma viremia. There was no correlation between the kinetics of viral rebound in plasma upon cessation of HAART and the size of the pool of the latent HIV reservoir prior to discontinuation of therapy. In addition, HIV env of the rebounding plasma virus was genetically distinct from either cell-associated HIV RNA or replication-competent virus within the pool of latently infected, resting CD4^{+} T cells in the majority of patients examined. These results underscore the possibility of existence of other persistent HIV reservoirs that could prompt rapid emergence of plasma viremia following cessation of HAART and the necessity to develop therapies directed toward such populations of infected cells.

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H. J. STELLBRINK*^{1}, J. VAN LUNZEN^{1}, M. WESTBY^{6}, E. O'SULLIVAN^{6}, N. CAMMACK^{6}, A. ADAM^{1}, L. WEITNER^{1}, B. KUHLMANN^{2}, C. HOFFMANN^{4}, H. HORST^{4}, S. FENSKE^{1}, G. SCHMIDT-HARTNACK^{1}, S. P. ARIES^{3}, K. DALHOFF^{3}, C. SCHNEIDER^{1}, O. DEGEN^{1}, F. T. HUFERT^{5}, K. TENNER-RACZ^{1}, and P. RACZ^{1}. Univ. Hosp. Eppendorf, Bernhard Nocht Inst. ^{1}Hamburg, ^{2}Hannover, ^{3}L˝beck, ^{4}Kiel, and ^{5}Freiburg, Germany and ^{6}Roche Welwyn, UK.

The study goal was to assess if IL-2 has a direct or indirect antiviral effect in vivo. The study was an open-label, randomized comparison of quadruple-drug HAART vs. HAART + IL-2 in 56 patients with >350/mm^{3} CD4+ T cells or recent seroconversion. Plasma viremia and T-cell subsets were determined at days 0,14,28, and monthly, after Tx discontinuation at weeks 1,2,4, and 8. Lymph node (LN) biopsies obtained before Tx and after >6 mths of neg. ultrasensitive RNA PCR were examined by in-situ hybridisation (n=31). 120 HIV-neg. controls were used to establish age- and gender-adjusted normal values for lymphocyte subsets.

At baseline: age 41, CD4+ 410, CD8+ 878 (/mm^{3}), CD4+:CD8+ ratio: 0.47, HIV RNA 4.83 log10, mean f/u: 572 days. The authors found that there was no difference in the magnitude or kinetics of plasma viremia decrease. IL-2 achieved a higher rate of normalisation of CD4+ T-cell counts, persisting after the end of IL-2 administration. Virus production was still detected in follow-up LN biopsies of 10/16 IL-2 and 11/15 non-IL-2 patients (35-40 cutting levels). Levels of proviral DNA and kinetics of decrease showed no significant difference between both groups. After Tx discontinuation, HIV RNA rebounded with very rapid kinetics and to transiently higher levels than at baseline. One IL-2 patient subsequently cleared viremia in the absence of therapy.

The authors concluded that despite a strong and durable immunomodulatory effect, IL-2 has no persistent effect on virus production or latent infection in vivo and the immunological control over HIV infection.

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J. MARTINEZ-PICADO^{1,2}, M. P. DePASQUALE^{1}, N. A. KARTSONIS^{1}, G. HANNA^{1}, J. WONG^{3}, D. FINZI^{4}, E. ROSENBERG^{1}, H. GUNTHARD^{3}, L. SUTTON^{1}, A. SAVARA^{1}, C. PETROPOULOS^{5}, N. HELLMANN^{5}, B. D. WALKER^{1}, D. D. RICHMAN^{3}, R. SILICIANO^{4}, and R. T. D'AQUILA*^{1}. ^{1}Massachusetts Gen. Hosp. and Harvard Med. Sch., Boston, MA; ^{2}Fndn. irsiCaixa, Barcelona, Spain; ^{3}Univ. of California, San Diego and VA Med. Ctr., La Jolla, CA; ^{4}Johns Hopkins Univ., Baltimore, MD; and ^{5}ViroLogic Inc., South San Francisco, CA.

Latent integrated HIV-1 can be reactivated in vitro from resting memory CD4+ T lymphocytes. Low level virus replication may continue in vivo even when plasma HIV-1 RNA is <50 copies/ml. Many patients have transient episodes of RNA >50 copies/ml. However, initial studies did not identify new drug resistance mutations in replication-competent HIV recovered in vitro from the blood latent reservoir during successful therapy (Wong, et al. & Finzi et al., Science 1997).

Viruses were isolated from PBMC during therapy which suppressed plasma HIV RNA to <50 copies/ml from 12 patients: 6 patients' viruses were biologically cloned (Finzi, et al.) and 6 pools of viruses isolated by Wong, et al. were molecularly cloned. Pol genotyping was by cycle sequencing and LiPA. Env C2-V3 was sequenced from biologic clones. Phylogenetic analyses (PHYLIP) and recombinant virus drug susceptibility assays (ViroLogic) were done.

Clonal sequencing analyses of replication-competent viruses recovered in vitro from latently infected blood lymphocytes identified new drug resistance mutations selected during successful therapy in 4 of the 5 drug-experienced, established infection patients with episodes of transient viremia. New resistance mutations did not develop in the 7 patients who did not have transient viremia. New resistance mutations were added to pre-existing mutational patterns in both protease (PR) and reverse transcriptase (RT) (two patients), only PR (one patient), or only RT (one patient). In one of these patients studied over 6 time points during successful therapy, PI-selected mutations accumulated in a virus sub-population and increasing resistance was confirmed by susceptibility assay. Phylogenetic analyses also showed drug selection pressure on protease, but not env. Repeated sampling over time, consistent findings in different patients, association with evidence of ongoing replication (env genetic diversity and transient viremia), and the specific mutation patterns observed suggest that the new resistance mutations were indeed selected during therapy.

The authors concluded that drug resistant HIV-1 can be selected in vivo in the absence of sustained rebound of viremia during treatment of established infection with regimens currently considered optimal.