1. HAIVN Harvard Medical School AIDS Initiative in Vietnam
ARV Drug Resistance
HAIVN
Harvard Medical School
AIDS Initiative in Vietnam

2. Learning Objectives At the end of this lecture, each trainee should:
Know how HIV develops resistance
Know factors increasing the risk for developing HIV resistance
Understand genotypic resistance testing
Learning Objectives

3. Outline of Presentation
What is resistance?
How does resistance develop?
Drug resistance in infants and children
Resistance testing
Lecture Content

4. What is HIV Drug Resistance?
Process by which changes (mutations) in the HIV virus allow the virus to become tolerant to antiretroviral drugs.
Resistance is the consequence of mutations that emerge in the viral proteins targeted by antiretroviral agents.
Definition of HIV drug resistance
Clavel and Hance. N Engl J Med 2004;350:

5. How does resistance develop?
Two important concepts:
HIV has an extremely high rate of virus replication and production.
The reverse transcription of viral RNA into DNA is prone to error (i.e mutations).
Two important concepts to understand.
1) HIV has an extremely high rate of virus replication and production. Up to 10 billion virions are produced each day.
2) The reverse transcription of viral RNA into DNA is very prone to error. On average one mutation is introduced for each viral genome produced - meaning that millions of billions of mutations can occur daily.
On average one mutation is introduced for each viral genome produced.

6. How does resistance develop?
Each patient, therefore, develops a mixture of viral ‘quasi-species’, each differing by one or more mutations.
These mutations may confer some selective advantage to the virus, such as a decrease in its susceptibility to an antiretroviral agent.
Potent combination ART prevents the development of resistant strains by maximally suppressing viral replication.
This description of how resistance develops may be easier to understand in graphic form – which is coming up in slide #8. So within each patient there are many virus strains which differ just slightly from each other (maybe by 1 or 2 mutations). Some of these virus strains will have a mutation which randomly confers tolerance or resistance to a particular ARV. This is why resistance can develop so quickly in some cases with mono-therapy. However, it is very very rare to have a virus strain with enough random mutations to have resistance to 2 or 3 drugs – which is why combination therapy is effective…..
Combination therapy can block the process of selecting resistant virus for two reasons.
-First, multiple mechanisms (each requiring different mutations) are required for resistance to occur to all drugs in the regimen.
-Second, multiple drugs suppress viral replication more effectively than single agents. In the absence of ongoing viral replication, the generation of new
variants is stopped.

7. How does resistance develop?
However, administration of antiretrovirals in an insufficiently potent manner selects for resistant strains.
Drug resistance emerges when HIV continues to replicate in the presence of levels of drugs that are insufficient to block viral replication but sufficient to exert a positive selective pressure on variants with decreased drug susceptibility.
In patients who receive HAART as a first line of antiretroviral therapy, the emergence of viral resistance is possible only if HIV continues to replicate in the presence of levels of drugs that are insufficient to block viral replication but sufficient to exert a positive selective pressure on variants with decreased drug susceptibility.

8. How does resistance develop?
This slide reviews the previous slides in graphic form.
Even before a patient takes ARV, there can be some mutant HIV with drug resistance. If ARV does not completely suppress viral reproduction, then resistant virus can continue to reproduce while “wild-type” or non-resistant virus is suppressed. Eventually, the resistant virus will become the predominant virus in the patient.

9. Reversion to Wild-Type Virus After Discontinuing ARV
After stopping ARV, the wild-type virus will become predominant, often within a few weeks. This is because the wild-type virus is more “fit” than the virus with mutations, which means that it reproduces more quickly. Therefore, as we will discuss late, resistance testing should be done while the patient is still on ARV.

10. Pediatric HIV Drug Resistance
HIV drug resistance occurs when:
When an infant acquires a resistant virus from the mother or
When resistance mutations develop because of ARV exposure for prophylaxis or treatment.
Infants and children can obtain HIV drug resistance for a number of reasons. Clarify that in the second scenario, the infant has acquired wild-type virus from the mother but then develops resistance subsequently due to inadequate ARV exposure.

11. HIV resistance: Perinatal Acquisition
ARV-naive mother infected with HIV already resistant to ARV
Mother exposed to ARVs before becoming pregnant
Mother exposed to ARVs during pregnancy (either for her own health or PMTCT)
Drug Resistant Virus
An infant can acquire resistant virus from the mother in utero, intrapartum or postpartum during breastfeeding.
The transmission of a resistant virus can occur:
From an ARV-naive mother infected with HIV already resistant to ARV
From a mother exposed to ARVs before becoming pregnant
From a mother who has been exposed to ARVs during pregnancy either for her own health or for prophylaxis of MTCT.
in-utero
intrapartum
postpartum during breastfeeding
Infant

12. HIV resistance: ARV Exposure
Infant or child with HIV
Exposure to subtherapeutic levels of ARVs during breastfeeding (i.e. from mothers receiving ART)
Infant ARV prophylaxis of MTCT intervention (i.e. single-dose NVP)
Treatment with ARVs (see next slides)
Infants and children may develop viral resistance as a result of:
Inadequate antiretroviral therapy
The infant ARV prophylaxis of MTCT intervention
Exposure to subtherapeutic levels of ARVs during breastfeeding (i.e. from mothers receiving ART). Exposure to subtherapeutic levels of ARVs during breastfeeding is mostly a theoretical concern.
Drug Resistant Virus

13. HIV resistance: ARV Exposure
Drug concentration in blood
Failed to take
medication
Changes of drug concentration in blood during treatment
Time
Missing a dose of medication can allow the blood level of the drug to fall below the level needed to fully suppress replication. Continued replication of HIV in the presence of low drug levels allows resistant virus to replicate and develop additional mutations. Over time, mutations accumulate and can create HIV which is resistance to multiple drugs.
Regular medication
Resistant HIV
Wild-type HIV
Lower limit of effective drug concentration in blood

14. HIV resistance: ARV Exposure
Social/Caregiver issues
Regimen issues
Poor potency
Toxicities
Wrong dose
Poor adherence
Host genetics
Poor absorption
Insufficient drug level
Insufficient drug levels in the blood allow virus to replicate in the presence of drug and resistance to develop. We focus on poor adherence as the cause of resistance, but there are other factors that can lead to low drug blood levels: poor quality drug, poor absorption of the drug from the gastrointestinal tract, increased metabolism and clearance of the drug, or interactions with other drugs.
Adherence in children is a special challenge because of factors relating to children, caregivers, medications and the interrelationships of these factors.
Specific problems that should be considered in treating children include the need to switch formulations as children cross thresholds of weight or age, and unavailability of a range of suitable pediatric ARV dose formulations.
Rapid clearance
Viral replication in the
presence of drug
Poor activation
Resistant virus
Drug interactions

15. Examples: Case 1
You wish to start an 8 month old infant male on antiretroviral therapy.
He was born to a mother who was diagnosed with HIV infection during labor.
The mother received no antiretroviral therapy for PMTCT.
The infant received single dose NVP only.
An case example designed to highlight the recommended treatment of an infant with HIV who received sdNVP for PMTCT.

16. What regimen should you start?
A) d4T / 3TC / NVP
B) AZT / 3TC / NVP
C) AZT / 3TC / ABC
D) AZT / 3TC / LPV-r (Aluvia)
The answer is D

17. Resistance acquired from infant PMTCT: single dose NVP
Lockman S, Shapiro RL, Smeaton LM, et al. Response to antiretroviral therapy after a single, peripartum dose of nevirapine. N Engl J Med 2007; 356: Thirty infants received NVP-based antiretroviral treatment (15 who received single-dose nevirapine at birth and 15 who received placebo). Virologic failure by the 6-month visit occurred in significantly more infants who had received a single dose of nevirapine than in infants who had received placebo (P<0.001).
N Engl J Med 2007; 356:

18. Children less than 12 months who have been exposed to NVP during PMTCT
Vietnam MOH Guidelines recommend:
Preferred regimen: AZT + 3TC + LPV/r
Alternative regimens:
a. d4T + 3TC + LPV/r
b. ABC + 3TC + LPV/r
c. AZT + 3TC + NVP or
d4T + 3TC + NVP
MOH guidelines

19. Resistance testing Types of resistance tests:
Genotypic testing: look for specific mutations in the genetic structure of the reverse transcriptase and protease enzymes that could cause drug resistance.
Phenotypic testing: measure the ability of a patient’s virus to grow in the presence of known concentrations of ARV. The test is very expensive and not available in Vietnam.

20. Genotypic Testing
The genetic code of the sample virus is compared to the wild type
The genetic code is a long chain of molecules called nucleotides
Each group of 3 nucleotides = “codon”, defines a particular amino acid used to build a new virus
The nucleotides in DNA and RNA code for amino acids to build proteins. Each group of 3 nucleotides codes for an amino acid. DNA and RNA each have 4 different nucleotides, so there are 64 possible codes (4 x 4 x 4). There are only 20 different amino acids, so several different codons can code for one amino acid. In addition, some codons signal the beginning or end of the amino acid sequence for a protein. Note: Lys: Lysine; Met: Methionine; Ser: Serine
Codon
AAA ATG AGC
Nucleotide
Lys
Met
Ser
Amino acid

21. Genotypic Testing
Mutations are described by a combination of letters and numbers, i.e.: M184V = 3TC resistance
M: Methionine is the name for the amino acid in the wild type virus
184: identifies the position of the codon
V: Valine is the name for the “changed” amino acid in the mutant sample
A mutation, or change in the nucleotide, will change the codon and the amino acid coded for by the codon.
Lys: Lysine
Met: Methionine
Ser: Serine
Codon 184 Mutation
Codon 184
AAA GTG AGC
AAA ATG AGC
Nucleotide
Lys
Lys
Met
Ser
Val
Ser
Amino acid

22. Genotypic Testing: Mutations Selected by nRTIs
Multi-nRTI Resistance: 69 Insertion Complex (affects all nRTIs currently approved by the US FDA)
3TC
d4T
Each ARV drugs selects for specific mutations that confer resistance to that drug. These are the mutations associated with first line NRTI.
AZT
IAS-USA

23. Genotypic Testing: Mutations Selected by NNRTIs and PI
EFV
NVP
Each ARV drugs selects for specific mutations that confer resistance to that drug. These are the mutations associated with the NNRTI and PI that we have in Vietnam.
Lopi/r
IAS-USA

24. Example of the genotype result from a child at NPH in Hanoi (test performed in HCMC)

26. Genotypic Testing: Limitations
The patient must be taking ARV at the time the test is done.
The test only detects mutations present in > 20% of circulating virus..
The viral load must be > 1,000 copies/ml.
Limitations of genotype testing.
The patient must be taking ARV with good adherence because if the patient stops ARV then the wild-type virus will become predominant (refer to slide#9 for explanation of this).
Mutations present in < 20% of the virus in the patient will not be detected.
In order for the test to be done in the laboratory, the VL must be greater than 1,000 copies/ml: at VL < 1,000 there is not enough virus for the test to be done.

27. Genotypic Testing: Limitations
Test is not widely available in Vietnam
Expensive (1,200,000 VND per test)
Genotype reports can be difficult to interpret in the setting of numerous resistance mutations.
Resistance testing is available in Hanoi and HCMC. At Pasteur Institute in HCMC, the cost is about 1,200,000 VND per test. The results can be difficult to interpret and apply in the clinical setting: it is best to consult with clinical experts.

28. Resistance Topics: Genetic Barrier
Genetic barrier is the number of mutations required to confer resistance to a drug.
Low genetic barrier: high level resistance with only one mutation
NVP, EFV: K103N
3TC: M184V
High genetic barrier: 3 or more mutations needed to develop high level resistance to most PI
The Genetic Barrier is a measure of how many mutations are needed to develop resistance to an ARV drug.

29. Resistance Topics: Cross-resistance
Cross-resistance within classes is common
Resistance to one NNRTI means resistance to all available NNRTIs
NVP and EFV
Resistance to one NRTI can indicate resistance to other NRTIs:
3TC and FTC
There is less cross-resistance among PI
Use the results of resistance testing to choose second-line drugs with the least risk for resistance
Cross resistance is universal among the 2 NNRTI (NVP, EFV) available in Vietnam: resistance to one confers resistance to the other. Cross resistance is also important when choosing an NRTI for second line regimens. Cross-resistance is less common among the PI.

30. Common mutations in patients with treatment failure on 1st line ARV
Selected by
Effects on ARV
M184V
3TC, FTC
- High resistance to 3TC
- ↓ viral fitness (HIV replicates more slowly)
- Delayed TAMS and ↑ susceptibility to AZT, d4T, TDF
TAMs
AZT, d4T
M41L, D67N, K70R, L210W, T215Y, K219Q
Resistance to all NRTIs based on number of TAMs
More TAMS = more resistance
Q151M, T69ins
AZT/ddI, ddI/d4T
Resistance to all NRTIs
T69ins: TDF resistance
K65R
TDF, ABC, ddI
- Variable resistance to TDF, ABC, ddI, 3TC
- ↑ susceptibility to AZT
K103N, Y181C
NVP, EFV
High resistance to both NVP and EFV
Review of common mutations seen after 1st line ARV treatment failure. Depending on the level of the audience, the lecturer could review this slide in detail or skip it altogether.

31. When to do resistance testing
Indications for ordering test in Viet Nam:
(ALL criteria should be met)
Patient must be currently taking ARV with good adherence for at least 6 months on the same regimen
Evidence for treatment failure (by clinical, immunological, and/or virological criteria)
Viral load testing done first and result > 1,000
More than one 2nd Line ARV regimen available
Resistance testing is not recommended by the Vietnam MOH. However, it can help in guiding the choice of second line treatment after failure of first line ARV. If 2nd line ARV is not available, or only one second line regimen is available, then resistance testing is not necessary.

32. Example: Case #2
A 3 year old boy whose mother recently died after a prolonged illness presents to the OPC.
He has been on ART x 1.5 years with d4T, 3TC, NVP.
Treatment failure was suspected based on decreased CD4 counts (immunological) and a failure to gain weight (clinical).
A viral load was performed and was copies/mL.

33. Example: Case #2
What are some reasons why he might have developed treatment failure?
If you did a genotype what type of resistance might you expect to see?
What would you choose for 2nd line therapy?
Questions to generate discussion:
What are some reasons why he might have developed treatment failure?: Participants might mention the need to know what the PMTCT regimen that the mother and child received. The boys mother recently died so we don’t know the caregiver situation for this child, but this could be a reason for poor adherence leading to treatment failure.
If you did a genotype what type of resistance might you expect to see?: Don’t expect participants to know the exact mutations but at least they should mention resistance to 3TC, d4T/AZT, and NNRTIs.
What would you choose for 2nd line therapy?: Can wait for genotype but likely standard 2nd line regimen – TDF/3TC/Aluvia – will be indicated.

34. Case 2: Genotype Drug Name NRTI Mutations Conclusion ZDV
D67N, K70R, M184V, K219Q
High-Level Resistance
3TC/
FTC
ddI
Low-Level Resistance
D4T
ABC
TDF
Possible Low-Level Resistance
The patient has multiple mutation NRTI mutations. These mutations follow the TAM pathway (67N, 70R,219Q) conferring resistance to AZT and d4T, and lower level resistance to ddI and ABC. M184 confers resistance to 3TC. TDF is the NRTI least affected

35. Case 2: Genotype
NNRTI:
Drug Name
Mutations
Conclusions
EFV
Y181C, Y188L
Resistant
NVP
PI: IDV, SQV/r, NFV, LPV/r, ATV/r, TPV/r, fosAPV/r:
No mutations detected
All PI sensitive
The NNRTI mutations confer high-level resistance to both NVP and EFV.
There are no PI mutations (which we expect because the patient has no history of PI use).

36. Key Points
Resistance occurs (1) when an infant acquires a resistant virus from the mother or (2) develops in the setting of an inadequately suppressive ARV therapy
When prescribing an ARV regimen, it is important to know a child’s prior ARV exposure (i.e. PMTCT regimen)
Dose ARVs carefully; remember to switch formulations as children cross thresholds of weight or age
Monitor and optimize adherence; Educating patients and caregivers about resistance may promote better adherence
Key Points

37. Key Points
Monitor closely for ARV effectiveness and signs of treatment failure.
For the patient who is failing therapy: check adherence issues first
Testing for HIV resistance to ARVs has become an important component of clinical care
Resistance assays can assist the clinician in selecting a maximally effective second line ARV regimen

38. Thank You
Questions?
Questions?