PRINCIPLES and TRENDS in MANAGEMENT of HIV DISEASE: PROBLEMS OF DRUG RESISTANCE in VIRUSES of DIFFERENT SUBTYPES

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1 PRINCIPLES and TRENDS in MANAGEMENT of HIV DISEASE: PROBLEMS OF DRUG RESISTANCE in VIRUSES of DIFFERENT SUBTYPES Mark A. Wainberg McGill University AIDS Centre Jewish General Hospital Montreal, Quebec, Canada

2 The Need for Combination Therapy The error rate of the HIV reverse transcriptase enzyme is extremely high. Therefore, mutations at all loci within the viral genome occur constantly. There is constant mutagenesis and selection of escape variants in HIV-infected individuals, as seen through emergence of both immunological escape variants and drug-resistant viruses.

3 CONCLUSION All patients harbour different HIV variants that include all possible single drug resistance mutations. The likelihood of having two such mutations within a single virus is much reduced but also occurs. The likelihood of having three such mutations within a single virus is virtually impossible except in cases of transmitted drug resistance

4 CONCLUSIONS The only way to prevent HIV mutagenesis and the emergence of escape mutants is to completely shut down viral replication. This requires the use of multiple drugs simultaneously to block vral replication. Ideally, all such drugs should be fully active. Patients must be as adherent as possible to therapy in order to maintain high plasma and intracellular levels of antiviral drugs. Otherwise, the presence of sub-standard levels of ARVs will select for drug resistance. Care must be taken when changing drug regimens to ensure that full anti- retroviral drug pressure is maintained.

5 Evidence in support of the low genetic barrier for resistance for NNRTIs: 1. Development of the K103N mutation after single-dose use of NVP in MTCT studies. 2. Evidence for development of K103N and other NNRTI mutations after treatment interruption. This results in compromise regarding future use of NNRTIs.

6 3. Patients to whom NNRTI mutations have been transmitted have diminished responsiveness to NNRTIs that are employed in subsequent therapy. 4. Patients who have NNRTI mutations detected by ultrasensitive assays rather than genotyping may be more likely to fully express such mutations when NNRTI therapy is introduced.

7 Baseline Samples with Detectable Minority Mutations and Treatment Outcomes Sample ID Baseline Minority Mutations Regimen Outcome Treatment Week of Failure Bulk Genotype Mutations at Failure 1 M184V ABC -3TC+EFV Failure 48 Unk 2 M184V ABC -3TC+EFV Failure 12 M184V 3 K103N ABC -3TC+EFV Failure 8 K103N, M184V 4 K103N, M148V ABC -3TC+EFV Failure 8 K103N, M184V 5 K103N ZDV+3TC+EFV Failure 24 K103N 6 K103N ZDV+3TC+EFV Failure 48 Unk 7 Y181C ABC -3TC+EFV Failure 12 WT 8 Y181C ABC -3TC+EFV Success NA 9 K103N ABC -3TC+EFV Success NA Johnson et al., PLos Medicine :e158.

8 Fraction of Treatment Success or Failure Versus Presence of Detectable Minority Drug Resistance Mutations Mutation Status No detectable drug Resistance mutation Treatment Success (n = 221) 219 (99.1%) Treatment Failure (n = 95) 88 (92.6%) Minority drug Resistance mutation 2 (0.9%) 7 (7.4%) Johnson et al., PLos Medicine :e158.

9 Population-based surveillance shows transmission cascades of HIV-1 variants harbouring drug resistance

10 Pol gene sufficient to reconstruct transmission events RT and protease sequences are conserved A single dominant viral species is transmitted & persists Sequence clustering infers viral interrelationships

11 Clustering of PHI transmission events Non-B Subtypes (n=17) Non-B Subtypes (n=53) * 11 Clustered PHIs n=293 2 Nonclustered PHIs n= * 11 * Brenner B, et al, JID April, 2007

12 Time intervals for onward PHI:PHI transmission Cluster Interval (months) PHI / Cluster >5 PHI / Cluster Cluster Size 64 clusters (n=158) 12 ± 6 months 20 clusters (n=168) 15 ± 9 months

13 Drug resistance in clustered and nonclustered PHI, in relation to CI potential transmitters Drug class CI - Treated PHI Cluster PHI Unique P values Any NRTI 64.4% 3.8% 9.0% Any NNRTI 37.8% 10.2% 9.0% n.s Any PI 42.2% 2.4% 5.0% 0.092

14 Transmitted Drug Resistance according to drug class and in relation to clustering Any NRTI Unique 9.3% Cluster (<5 PHI) 3.8% Cluster (>5 PHI) 2.1% p *** 215 Revertants 5.8% 1.7% 0.4% *** M184V 2.1% 1.3% 0.4% Any NNRTI 6.7% 5.9% 15.5% ** G190A 1.1% 0.4% 10.5% *** Any PI 5.0% 3.8% 5.4% Major PI 2.9% 0.5% 0.4% * MDR 4.5% 1.7% 2.1% * Any resistance 14.3% 12.4% 20.5%

15 Age distribution of PHI in clustered and unique transmissions 60 CLUSTERS UNIQUE Nombre des cas Age

16 Temporal changes in clustering profile in the male PHI population (n=730) Cluster Year of diagnosis Profile 06/ / / / / / / /2007 Unique % 50.0% 49.3% 49.8% 50.0% PHI % 26.9% 23.7% 24.2% 25.6% Small clusters PHI (< 5 PHI/ cluster) Size 2.9 ± ± ± ± 0.1 % 23.0 % 26.9% 25.9% 24.3% Large clusters PHI ( 5 PHI/ cluster) Size 11.9 ± ± ± ± 1.0

17 Distribution of new diagnoses in Quebec where the last HIV negative test is known (INSPQ report ) Time from last HIV-1 negative test <1 year 1-2 years 2-3 years 3-4 years 4-5 years 6-10 years >10 years Females (n=64) 25.0% 17.2% 17.2% 10.9% 9.4% 18.8% 1.6% Males (n=518) 32.1% 22.6% 10.0% 9.1% 5.6% 13.9% 6.7%

18 The potential impact of rapid testing in HIV-1 prevention Disease stage at diagnosis ( ) Time from last reported HIV negative test at diagnosis % of Cases (n=518 cases) Non-specific illness 30 AIDS defining illness Chronic symptomatic 20 Recent / asymptomatic 10 Acute % of cases ( , n= 1484) Duration from last known HIV negative test (yrs)

19 Summary NNRTIs that are now approved for use in first-line therapy may be more fragile than some long-term clinical trials may suggest. It is only logical that the transmission of NNRTI mutations will continue to increase in settings in which NNRTIs have been used extensively in firstclass therapy. This problem is further exacerbated by data on clustering of NNRTI mutations in new transmissions, which reflects the high fitness of viruses that carry NNRTI mutations.

20 Global distribution of HIV-1 subtypes 1.3 million 2 million 4.8 million URF 4.2% AE 3.1% G 5% D 3.6% AG AG 6.7% 6.7% A 12% 12% B 10%

21 Do Differences Exist among HIV Subtypes in the Development of Drug Resistance?

22 Silent Mutation at Codon 106 responsible for the V106M mutation in clade C RT with NNRTIs HIV-1 RT Subtype B Subtype C Wild type codon at position 106 V(GTA) V(GTG) In clade C, V106M arises two codon changes M(ATG) In clade B, V106A occurs A(GCA) two codon changes

23 Marconi et al showed that V106M is the second most common NNRTI mutation after K103N in subtype C patients. In contrast, V106A/M is very rarely seen with subtype B isolates. Clinical Infectious Diseases 15:1589, 2008

24 Rapid Selection of K65R Resistance in Subtype C Isolates [TDF] (µm) K65R wt (wk 34-78) Subtype C Subtype B Week

25 History of 23 Botswana Patients Treated with ddi/d4t plus 3TC or NVP No. Patients 23 No. Patients failing 15 No. Patients with K65R 7 No. Patients with L74V 0

26 Resistance profile of patients failing First Line ART in Malawi when using Clinical and Immunologic Monitoring Abstract TUAB0105 M Hosseinipour, JJ van Oosterhout, R Weigel, J Nelson, S Fiscus, J Eron, J Kumwenda

27 Background The Malawi ART program scale-up: >150,000 patients started on d4t/3tc/nvp A substantial minority with have virologic failure and eventually clinical failure. In failing patients resistance will be present Few data from Africa on resistance patterns

28 Results 96 patients identified as ART Failure with VL>1000 copies 16 Clinical- WHO stage IV 87 Immunological- CD4 decline 66 on d4t/3tc/nvp 30 on ZDV/3TC/NVP due to d4t toxicity 2 samples did not amplify 94 samples for analysis

29 Expected Mutations M184V or M184I 81% NNRTI mutations 93% Median 2 (range 0-3) 181C 55%, 190A 30%, 103N 28% Wild Type Virus 5% M184 only 0% NNRTI mutations only 2% M184V & NNRTI mutations only 16%

30 Resistance Patterns % NNRTI mutations +/-184V containing virus + additional mutations TAM Containing Virus 56% Tenofovir mutations (K65R or K70E) 23% Tenofovir & TAM 7% Q151M Complex 19% Pan-Nucleoside Mutation Combinations Q151M Complex & Tenofovir mutations 16% 69 insertion 1% Pan-Nucleoside 17% (Q151 & TDF associated mutations or 69 insertion)

31 Similar findings have now been obtained in Capetown,South Africa by C. Orrell et al working with subtype C infected populations

32 Rationale Does the nucleotide sequence trigger an increased probability of the development of the K65R resistance mutation in subtype C? Subtype B 5'-... GCC ATA AA G AAA AAA GAC AGC...-3' 3'-... CGG TAT TT C TT T TTT CTG TCG...-5' A I K K K D S... Subtype C 5'-... GCC AT A AAA AAG AAA GAC AGC...-3' 3'-... CGG TAT TT T TT C TTT CTG TCG...-5' A I K K K D S... 5'-... GCC ATA AA G A GA AAA GAC AGC...-3' 3'-... GCC TAT TT C T CT TTT CTG AGC...-5' A I K R K D S... 5'-... GCC ATA AA A A GG AAA GAC AGC...-3' 3'-... CGG TAT TT T T CC TTT CTG TCG...-5' A I K R K D S... Biochemical analysis of the syntheses of (-)strand DNA from viral RNA and (+)strand DNA from viral (-)strand DNA by the RTs of subtypes B and C.

33 (+)strand DNA synthesis (1) Only the subtype C sequence triggers a pausing site that increases the probability of a nucleotide misincorporation event which in turn leads to the K65R mutation.

34 (+)strand DNA synthesis (2) The pausing patterns are driven by the nucleotide templates and are independent of the RT enzymes used (subtype B vs C).

35 Validation in cell culture What is the propensity of different recombinant viruses to develop the K65R resistance mutation under N(t)RTI treatment? AAG AAA AAA AAA AAA AAA AAG AAG AAA AAA AAG AAA...-3 NL4-3 (wt) NL4-3 (K64K) NL4-3 (K65K) NL4-3 (K64K/K65K) G K65R Infections of MT-2 cells and CBMCs with these viruses followed by treatment with different N(t)RTIs (single drugs or in combination).

36 Drugs Selections in CBMCs Virus NL4-3 (wt) NL4-3 (64/65) Mutation Week Mutation Week TFV none >35 K65R 25 TFV + 3TC M184V 15 K65R 20 NL4-3 (wt) 5'-... AA G AA A...-3' 3'-... TT C TT T...-5' K K... Subtype B sequence NL4-3 (64/65) 5'-... AA A AA G...-3' 3'-... TT T TT C...-5' K K... Subtype C sequence at positions 64/65 in a subtype B backbone The double mutant NL4-3 (64/65) acquires K65R more rapidly in CBMCs than wild-type NL4-3.

37 Mutations in CBMCs after 20 Weeks DRUG NL4-3 (wt) VIRUS NL4-3 (64/65) ddi None L74V TNF None K65R TNF/3TC M184V K65R

38 Mutations in MT-2 Cells after 10 Weeks DRUG NL4-3 (wt) VIRUS NL4-3 (64) NL4-3 (65) NL4-3 (64/65) 3TC M184I Not done Not done M184I FTC M184I M184I M184I M184I ABC M184I M184I M184I K65R ddi L74V M184I V75I K65R d4t None None None K65R TNF None None None K65R

39 Conclusion K65R will be a more important mutation in subtype C than subtype B viruses. This may affect both prevention research strategies as well as treatment options over time in areas in which subtype C viruses are predominant

40 WHAT ABOUT PRE-EXPOSURE PROPHYLAXIS (PREP)? Clinical trials are underway in regard to the combination of TDF/FTC (Truvada). Will some individuals who opt for PREP be infected and not know it, ie be positive for HIV by PCR but negative by Ab test? Will this select for K65R in subtype C populations? This highlights the need for cheap, qualitative PCR-based diagnostic tests for HIV, perhaps by DBS

41 General Considerations in Drug Resistance re Prevention Research The male partner is infected with a drugresistant variant of HIV-1. Can the presence of transmissible drug-resistant viruses in a population overcome whatever ARV block is in place through use of a microbicide or PREP? There is scant data on this topic.

42 CONCERNS RE MICROBICIDES A woman is HIV-infected. She either does not know it or does but chooses to use ARVs to protect a sexual partner. Will drug resistance be selected? This will depend in large part on whether the microbicide is systemically absorbed and exerts the equivalent of monotherapy. But, inadequate absorption or only very low level absorption may be inadequate to apply antiviral drug pressure and select for drug resistance. e.g. UC-781, TMC-120

43 Higher Tendency of Subtype C Viruses to Select for K65R? There is no evidence that three good drugs used in tandem will not be able to suppress viral replication in efficient fashion, regardless of viral subtype. It is unknown whether failure to be adherent to ARVs will select K65R faster in subtype C viruses but this is a concern. Durability of regimens that include N(t)RTIs that may select K65R in subtype C populations seems good although only limited data are available.

44 Sexual Transmission of Drug Resistance Mutations Approximately 5-10% of all new HIV infections in developed countries now include at least one drug-resistance related mutation. Transmitted drug resistance is now increasingly being reported in developing countries. No information is yet available on whether K65R may be sexually transmitted.

45 CONCLUSION HIV genotyping should be performed on all patients prior to prescription of ARVs HIV genotyping should be performed on all patients prior to switching to a second-line regimen. The above considerations are not yet practical in terms of being implemented in many developing country settings.

46 Sexual Transmission of HIV Drug Resistance It is important to give support to the WHO surveillance programme for HIV drug resistance (HIVRESNET). Transmission of HIV drug resistance is of obvious concern in regard to public health. Donor agencies should recognize that drug resistance is an inevitable consequence of the use of ARVs and should provide funds for HIV resistance monitoring at the same time that they provide ARVs.

47 T H A N K Y O U