Emergence of HIV-1 mutated strains after interruption of highly active antiretroviral therapy in chronically infected patients
Daniel, Nathaliea,b; Schneider, Veroniquec; Pialoux, Gillesd; Krivine, Annee; Grabar, Sophief; Nguyen, Thu Huyend; Girard, Pierre-Marieg; Rozenbaum, Willyd; Salmon, Dominiquea
Departments of aInternal Medicine, eVirology, and fBiostatistics, Cochin Hospital, Paris, France; bDepartment of Immunology, Cochin Institut, INSERM U567, CNRS UMR 8104, Rene Descartes University, Paris, France; Departments of cVirology, and dInfectious Diseases, Tenon Hospital, Paris, France; and gDepartment of Infectious Diseases, Saint Antoine Hospital, Paris, France.
The first two authors contributed equally to this work.
Sponsorship: This research was funded by ARVIH. CEMIT and SIDACTION provided grant support for N.D.
Received: 4 March 2003; revised: 29 March 2003; accepted: 28 April 2003.
We report the emergence of HIV-1 mutated strains after long-term viral suppression in four patients who interrupted highly active antiretroviral therapy (HAART). In two cases, mutations pre-existed in proviral DNA before HAART interruption. All mutations were associated with resistance to nucleoside reverse transcriptase inhibitors, and three of the four patients had prolonged periods of monotherapy or dual therapy. Resistant strains may re-emerge rapidly even in patients harbouring a majority of wild-type virus in proviral DNA before treatment interruption.
In patients with undetectable viral loads and high CD4 T-cell counts, the objectives of structured treatment interruptions are to reduce the toxicity and cost of highly active antiretroviral therapy (HAART). However, in such interruptions, the risk of the re-emergence of HIV-1 mutated strains is not well established. Our objective was to study the emergence of mutated strains in HIV-1 chronically infected patients with undetectable viraemia after HAART interruption, and to investigate whether these mutations were present in proviral DNA before HAART interruption.
The 17 patients studied were previously included in the LIPTHERA vaccine study, an HIV lipopeptides vaccine trial . The reverse transcriptase (RT) and the protease sequences were not included in the vaccine. Immunizations were followed by HAART interruption 24 weeks later, and to avoid transient monotherapy, non-nucleoside reverse transcriptase inhibitors (NNRTI) were stopped one week before the other drugs.
HIV-1-RNA genotyping was performed 4 and 8 weeks after HAART interruption from plasma samples (RNA was then reverse transcribed to complementary DNA). On the day of interruption (D0) proviral DNA genotyping was performed as the viral load was less than 50 copies/ml. Proviral HIV DNA was extracted from the peripheral blood mononuclear cells of patients. Both were genotyped for the RT and protease genes. The RT and protease genes were amplified as previously described , and polymerase chain reaction products were sequenced using the ABI Prism Big Dye Primer sequencing kit (ABI, Applied Biosystems, Foster City, CA, USA). Only mutations known to be associated with antiretroviral resistance were considered .
At D0, patients had been on HAART for a median of 3.6 years (2.3–5.8). In four out of 17 patients (23.5%), mutated strains were identified in the plasma 4 or 6 weeks after interruption (Table 1). All evidenced resistance mutations were located in the polymerase gene and were associated with resistance to nucleoside reverse transcriptase inhibitors. No mutation associated with resistance to protease inhibitors or to NNRTI was found. In two patients (R03B and R10N), the mutations found in plasma HIV-1 RNA after rebound were similar to those in proviral DNA before HAART interruption. Regarding the treatment regimens, patient R10N was treated for 10 years with 4.4 years of monotherapy or dual therapy, whereas patient R03B always received the same effective HAART. In the two remaining patients (C03B and C06F), the mutations in plasma HIV-1 RNA were not seen in proviral DNA before HAART interruption. These patients also had a therapeutic exposure (5.3 and 6.7 years, respectively) with 2 years of monotherapy or dual therapy. Two patients (R11L and C12J), with wild-type strains in the plasma after rebound, harboured resistance mutations in proviral DNA before HAART interruption. Of the 10 patients (62.5%) with HIV-1 wild-type strains before and after HAART interruption, only five had previously been exposed to monotherapy or dual therapy.
Most structured treatment interruption studies did not detect any mutations during rebound [4–7]. Nevertheless, Schweighardt et al.  reported the emergence of HIV-1 mutated strains (mainly K101E and K103N mutations) but related to transient monotherapy with NNRTI.
Four of our patients harboured mutated strains at rebound and three of them had had long previous monotherapy or dual therapy. This could explain the constitution of a pool of mutations in different reservoirs . Moreover, in patients without such previous exposure, a residual HIV-1 replication may occur , even on HAART, with the transcription of HIV messenger RNA and undetectable viraemia . In such cases, mutated strains could be selected and emerge with treatment interruption .
In two cases, proviral DNA genotyping allowed us to find mutations evidenced in plasma HIV-1 RNA. However, in patients with mutated HIV-1-RNA strains in plasma after rebound and wild-type strains in proviral DNA before HAART interruption, several hypotheses could be proposed. First, mutated strains could be harboured in reservoirs other than resting CD4 T cells, as suggested by Chun et al. . Indeed, Finzi et al.  did not find mutations in the resting CD4 T cells of chronically infected patients on HAART for more than 30 months. In some reservoirs such as the cerebrospinal fluid  and female genital tract  the concentration of antiretroviral drugs is suboptimal, which could allow the selection of mutated strains, whereas plasma strains remain wild. Second, in our study, proviral DNA genotyping was based on direct sequencing, which only detects major strains. The detection of a wild-type strain using this technique does not exclude the presence of minority mutated strains. However, in the study of Martinez-Picado et al.  in which proviral DNA genotyping was based on clonal sequencing, the M184V mutation detected in plasma HIV-1 RNA after several structured treatment interruptions was not identified in proviral DNA before each structured treatment interruption.
In our study, two patients had a wild-type strain at rebound in spite of mutations in proviral DNA. When HAART is interrupted, wild-type and mutated strains are competing. The initial doubling time of mutated strains is similar to that of wild-type strains, but off HAART the fitness of mutated strains decreases rapidly . The speed of re-emergence of wild-type strains probably depends not only on the fitness but also on the proportion of mutated and wild populations. In patient C06F a mutated strain was observed 4 weeks after HAART interruption, and the wild-type strain became the majority soon after 8 weeks. This is in accordance with another observation that showed that mutated strains often disappeared 6–8 weeks after HAART interruption .
In conclusion, one must be cautious with structured treatment interruptions in patients with previous antiretroviral monotherapy or dual therapy, given the risk of the re-emergence of resistant strains after HAART interruption. In chronically infected patients with undetectable viraemia, peripheral blood mononuclear cells could harbour mutations in proviral DNA. However, the initial detection of mutated strains in proviral DNA can be followed by a plasma rebound with wild-type strains, and conversely, the absence of mutation in proviral DNA does not exclude a plasma rebound with mutated strains. Proviral DNA genotyping before HAART interruption does not therefore seem sensitive enough routinely to evaluate the risk of resistant strain re-emergence.
1. Salmon D, Daniel N, Pialoux G, Schneider V, Krivine K, Charmeteau B, et al. CD4+ and CD8+ T-cell responses are induced in chronically HIV-1 infected patients after immunization by a lipopeptide vaccine. In: 10th Conference on Retroviruses and Opportunistic Infections, session 78. Boston, USA, 11–14 February 2003 [Poster 643].
2. Pasquier C, Millot N, Njouom R, Sandres K, Cazabat M, Puel J, et al. HIV-1 subtyping using phylogenetic analysis of pol gene sequences. J Virol Methods 2001, 94:45–54.
3. Hirsch MS, Brun-Vezinet F, D'Aquila RT, Hammer SM, Johnson VA, Kuritzkes DR, et al. Antiretroviral drug resistance testing in adult HIV-1 infection: recommendations of an International AIDS Society – USA Panel. JAMA 2000, 283:2417–2426.
4. Garcia F, Plana M, Vidal C, Cruceta A, O'Brien WA, Pantaleo G, et al. Dynamics of viral load rebound and immunological changes after stopping effective antiretroviral therapy. AIDS 1999, 13:F79–F86.
5. Ruiz L, Martinez-Picado J, Romeu J, Paredes R, Zayat MK, Marfil S, et al. Structured treatment interruption in chronically HIV-1 infected patients after long-term viral suppression. AIDS 2000, 14:397–403.
6. Garcia F, Plana M, Ortiz GM, Bonhoeffer S, Soriano A, Vidal C, et al. The virological and immunological consequences of structured treatment interruptions in chronic HIV-1 infection. AIDS 2001, 15:F29–F40.
7. Carcelain G, Tubiana R, Samri A, Calvez V, Delaugerre C, Agut H, et al. Transient mobilization of human immunodeficiency virus (HIV)-specific CD4 T-helper cells fails to control virus rebound during intermittent antiretroviral therapy in chronic HIV type 1 infection. J Virol 2000, 75:234–241.
8. Schweighardt B, Ortiz GM, Grant RM, Wellons M, Miralles GD, Kostrikis LG, et al. Emergence of drug-resistant HIV-1 variants in patients undergoing structured treatment interruptions [Letter]. AIDS 2002, 16:2342–2344.
9. Wong JK, Hezareh M, Gunthard HF, Havlir DV, Ignacio CC, Spina CA, et al. Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science 1997, 278:1291–1295.
10. Appay V, Hansasuta P, Sutton J, Schrier RD, Wong JK, Furtado M, et al. Persistent HIV-1-specific cellular responses despite prolonged therapeutic viral suppression. AIDS 2002, 16:161–170.
11. Chun TW, Davey RT Jr, Ostrowski M, Shawn Justement J, Engel D, Mullins JI, et al. Relationship between pre-existing viral reservoirs and the re-emergence of plasma viremia after discontinuation of highly active anti-retroviral therapy. Nat Med 2000, 6:757–761.
12. Finzi D, Hermankova M, Pierson T, Carruth LM, Buck C, Chaisson RE, et al. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 1997, 278:1295–1300.
13. Cunningham PH, Smith DG, Satchell C, Cooper DA, Brew B. Evidence for independent development of resistance to HIV-1 reverse transcriptase inhibitors in the cerebrospinal fluid. AIDS 2000, 14:1949–1954.
14. Si-Mohamed A, Kazatchkine MD, Heard I, Goujon C, Prazuck T, Aymard G, et al. Selection of drug-resistant variants in the female genital tract of human immunodeficiency virus type 1-infected women receiving antiretroviral therapy. J Infect Dis 2000, 182:11:112–122.
15. Martinez-Picado J, Morales-Lopetegi K, Wrin T, Prado JG, Frost SD, Petropoulos CJ, et al. Selection of drug-resistant HIV-1 mutants in response to repeated structured treatment interruptions. AIDS 2002, 16:895–899.
16. Birk M, Svedhem V, Sönnerborg A. Kinetics of HIV-1 RNA and resistance-associated mutations after cessation of antiretroviral combination therapy. AIDS 2001, 15:1359–1368.
17. Deeks SG, Wrin T, Liegler T, Hoh R, Hayden M, Barbour JD, et al. Virologic and immunologic consequences of discontinuing combination antiretroviral-drug therapy in HIV-infected patients with detectable viremia. N Engl J Med 2001, 344:472–480.#m AcknowledgementsThe authors would like to thank Vincent Calvez and Anne-Genevieve Marcelin for confirmation of the results of HIV-1 proviral DNA genotyping, Jean-Paul Viard for helpful discussions, and the patients of the Cochin and Tenon Hospitals who provided blood samples.
© 2003 Lippincott Williams & Wilkins, Inc.