Cattedra di Malattie Infettive e Tropicali, Dipartimento di Scienze Cliniche “Luigi Sacco”, Università degli Studi, Milan, Italy
Supported by AIDS research grants (III, IV, and V AIDS Projects: grants 40C.80, 40D.74, and 30F.46) from the Istituto Superiore di Sanità, Rome to S. Rusconi, “Progetto Giovani” Ministero Università e Ricerca Scientifica Tecnologica 2001 to E. Bulgheroni, and Anlaids ONLUS Sez. Lombarda, Milan, Italy. M. Lo Cicero is the recipient of a research fellowship provided by the Fondazione Andrea e Libi Lorini, Milan, Italy.
Presented in part at the Seventh International Congress on Drug Therapy in HIV Infection, Glasgow, UK, November 14-18, 2004 (poster 142).
To the Editor:
Tenofovir DF (TDF) is a new acyclic nucleotide inhibitor used to treat HIV-infected patients; because of its resistance profile, it is suitable for treating cases of therapeutic failure. Furthermore, it is used on subjects in whom primary HIV infection is attributable to the transmission of resistant strains as well.1 Patients with a high degree of resistance to nucleoside reverse transcriptase inhibitors (NRTIs) present with different mutations in the reverse transcriptase (RT) gene, particularly the thymidine analogue mutations (TAMs) selected after treatment with zidovudine (ZDV) and stavudine (d4T)2-4 or Q151M multidrug resistance (MDR) complex. All these mutations have led to cross-resistance to 6 NRTIs,5 but they have not reduced susceptibility to TDF. To the contrary, mutations like K65R (attributable to therapy with TDF but also selected by abacavir, didanosine, and zalcitabine) and T69S insertions are rare (<2%) in pretreated subjects.6,7 In addition, the M184V mutation that is selected after treatment with lamivudine8 seems to increase TDF sensitivity, reducing the resistance attributable to the presence of the K65R mutation.9
This study was aimed at evaluating the appearance of mutations (known or new) not previously associated with drug resistance. We analyzed genotypic resistance profiles during therapy as well as premature virologic failures.
Fifteen HIV-1-infected patients were selected for participation in the Tenofovir Expanded Access Program at our institute. These patients had experienced multiple failures after different kinds of combination therapies. The patients, excluding 2 subjects, were treated with at least 3 drugs at the time of enrollment. Before TDF treatment, these patients had HIV-1 plasma levels (HIV RNA) from <50 to 274,000 copies/mL. All these patients received TDF for 1 year and had been checked for their clinical status and monitored for hematology and biochemistry parameters, CD4 cell count, and HIV RNA level at different time points (at baseline and after 6 and 12 months), including eventual rebounds (ie, increase ≥1 log10 compared with the lowest value). Viral genotyping was performed at all time points on plasma samples.
Twelve patients showed various degrees of virologic response, and it was possible to divide them into 2 groups: responders and nonresponders. In 9 cases, TDF led to a decrease of the HIV RNA level independently from that at baseline, and in 78% of these cases, virologic suppression was below 50 copies/mL. The remaining cases showed a decrease but with an HIV RNA value >50 copies/mL. In 3 cases, TDF treatment did not lead to any significant reduction in HIV RNA values during the 1 year of follow-up. After 6 months of TDF, HIV RNA levels had risen in each of the nonresponders.
With respect to the genotype analysis, almost all patients showed a large mutation number at baseline because of previous experience with multiple antiretroviral regimens. Our patients presented with at least 5 mutations or more before TDF treatment, excluding subjects TDF04, TDF07, and TDF09. In particular, all patients had at least 1 TAM at position 41, 67, 70, 210, 215, or 219, and there were 3 or more TAMs in 67% of our subjects. Moreover, many patients (59%) had the M184V mutation (Table 1).
In 8 of 9 responders, it was not possible to determinate any mutation during the follow-up period because HIV-RNA values were below 50 copies/mL (limit of quantification for our test) and RNA extraction was not successful. In nonresponder patients, there was the presence of at least 4 TAMs when virologic suppression was not evident, and the most frequent pattern was the change at codons 41, 67, 210, and 215, often followed by the appearance of a fifth TAM during the follow-up period. Finally, 2 of 3 nonresponders showed an absence of virologic suppression with various degrees of rebound in HIV RNA levels. In 2 cases (TDF10 and TDF12), the HIV RNA rebound corresponded with the disappearance of the M184V mutation and the persistence of at least 4 TAMs (Table 2).
In our study, most of the patients demonstrating multiple failure who received TDF presented a response to the antiretroviral therapy, including TDF, and in almost all subjects, viral suppression was below the limit of detection (<50 copies/mL). This level of suppression was not possible in only 25% of treated subjects. In all patients in whom the genotypic sequence was determined, a TAM 1 profile was evidenced, which was characterized by a change at RT amino acids 41, 210, and 215.10,11 Between responders and nonresponders, there was a different number of TAMs; a fourth or a fifth TAM was often present. This finding would explain the different virologic responses among the patients-an increasing number of TAMs were present, together with a gradual loss of HIV-1 suppression and a reduction of treatment response. Another study indicated that mutations at positions L210W and K219Q/E appeared later during treatment with NRTIs, suggesting that the presence of 2 mutations among codons 41, 67, 70, and 215 would affect viral replicative capacity.12 In our study, the K65R mutation did not appear in any patient. Clinical studies have shown that the K65R mutation occurred in approximately 3% of subjects after up to 96 weeks of TDF therapy.12-14 Moreover, this mutation showed no compatibility with the presence of TAMs, as has been demonstrated previously in vitro15 and in vivo.16 A diminished replicative capacity has been described in HIV-1 isolates containing K65R and M184V9,17 or alone.18 With regard to the M184V mutation, this amino acid change is selected after treatment with lamivudine and its presence influences replicative capacity.19 This finding was confirmed by our data regarding 2 of 3 nonresponder patients. In both cases, the increased HIV RNA value was coincidental to the outgrowth of the M184V mutation. Treatment with TDF did not induce any new TAMs over a 12-month period in this cohort of multiexperienced patients when compared with the genotypic profile at baseline. This feature underlines the high genetic barrier possessed by TDF and confirms that this compound has an important role in HIV therapy as a first-line drug as well as in salvage regimens.
Mirko Lo Cicero, MS
Elisabetta Bulgheroni, PhD
Francesca Soster, MS
Ottavia Viganò, MD
Paola Cicconi, MD
Massimo Galli, MD
Stefano Rusconi, MD
Sezione di Malattie Infettive e Tropicali Dipartimento di Scienze Cliniche “Luigi Sacco” Università degli Studi Milan, Italy
1. Miller V, Larder BA. Mutational patterns in the HIV genome and cross-resistance following nucleoside and nucleotide analogue drug exposure. Antivir Ther
. 2001;6(Suppl 3):25-44.
2. Larder BA, Kemp SD. Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance to zidovudine (AZT). Science
3. Kellam P, Boucher CAB, Larder B. Fifth mutation in human immunodeficiency virus type 1 reverse transcriptase contributes to the development of high-level resistance to zidovudine. Proc Natl Acad Sci USA
4. Harrigan PR, Konghorn I, Bloor S, et al. Significance of amino acid variation at human immunodeficiency virus type 1 reverse transcriptase residue 210 for zidovudine susceptibility. J Virol
5. Shirasaka T, Kavlick MF, Ueno T, et al. Emergence of human immunodeficiency virus type 1 variants with resistance to multiple dideoxynucleosides in patients receiving therapy with dideoxynucleosides. Proc Natl Acad Sci USA
6. Larder BA, Bloor S, Kemp SD, et al. A family of insertion mutations between codon 67 and 70 of human immunodeficiency virus type 1 reverse transcriptase confer multinucleoside analogue resistance. Antimicrob Agents Chemother
7. Harrigan PR, Miller MD, McKenna P, et al. Phenotypic susceptibilities to tenofovir in a large panel of clinically derived human immunodeficiency virus type 1 isolates. Antimicrob Agents Chemother
8. Bloor S, Kemp SD, Hertogs K, et al. Patterns of HIV drug resistance in routine clinical practice: a survey of almost 12000 samples from the USA in 1999. Antivir Ther
. 2000;5(Suppl 3):169.
9. Deval J, White KL, Miller MD, et al. Mechanistic basis for reduced viral and enzymatic fitness of HIV-1 reverse transcriptase containing both K65R and M184V mutations. J Biol Chem
10. Flandre P, Descamps D, Joly V, et al. Predictive factors and selection of thymidine analogue mutations by nucleoside reverse transcriptase inhibitors according to initial regimen received. Antivir Ther
11. Flandre P, Descamps D, Joly V, et al. A survival method to estimate the time to occurrence of mutations: an application to thymidine analogue mutations in HIV-1-infected patients. J Infect Dis
12. Margot NA, Isaacson E, McGowan I, et al. Genotypic and phenotypic analyses of HIV-1 in antiretroviral-experienced patients treated with tenofovir DF. AIDS
13. Margot NA, Isaacson E, McGowan I, et al. Extended treatment with tenofovir disoproxil fumarate in treatment-experienced HIV-1-infected patients: genotypic, phenotypic, and rebound analyses. J Acquir Immune Defic Syndr
14. Miller MD, Margot N, Lu B, et al. Genotypic and phenotypic predictors of the magnitude of response to tenofovir disoproxil fumarate treatment in antiretroviral-experienced patients. J Infect Dis
15. Parikh U, Koontz D, Hammond J, et al. K65R: a multi-nucleoside resistance mutation of low but increasing frequency. Antivir Ther
16. Winston A, Pozniak A, Mandalia S, et al. Which nucleoside and nucleotide backbone combinations select for the K65R mutation in HIV-1 reverse transcriptase. AIDS
17. Deval J, Navarro JM, Selmi B, et al. A loss of viral replicative capacity correlates with altered DNA polymerization kinetics by the human immunodeficiency virus reverse transcriptase bearing the K65R and L74V dideoxynucleoside resistance substitutions. J Biol Chem
18. Weber J, Chakraborty B, Weberova J, et al. Diminished replicative fitness of primary human immunodeficiency virus type 1 isolates harboring the K65R mutation. J Clin Microbiol
19. Wainberg MA, Drosopoulos WC, Salomon H, et al. Enhanced fidelity of 3TC-selected mutant HIV-1 reverse transcriptase. Science