1. ARV failure and resistance for the paediatrician
Douglas Watson, M.D.
University of Maryland
11 December 2013

2. REVERSE TRANSCRIPTASE INHIBITORS (NRTIs & NNRTIs)
Integrase inhibitors
Reverse transcriptase inhibitors (nucleoside and non-nucleoside RT inhibitors) prevent the synthesis of a DNA copy of the viral RNA genome. Protease inhibitors act on the virus after it buds through the cell membrane, inhibiting the cutting of long inactive polypeptide chain into shorter functional proteins that the virus needs to infect the next cell. As our bodies do not perform reverse transciption, inhibiting that step should not adversely affect us. But many of the HIV nucleoside reverse transcriptase inhibitors do have an effect on the enzyme that replicates mitochondrial DNA- accounting for the toxicity of the NRTIs. There is one fusion inhibitor in clinical use, but it is extremely expensive to make. More useful integrase inhibitors show promising early results in humans.
Fusion Inhibitors
PROTEASE INHIBITORS
Attachment Inhibitors
HIV DRUG TARGETS

3. Antiretrovirals in the U.S.- 2013
Nucleoside analogues (NRTIs)
Zidovudine (ZDV, AZT, Retrovir)
Lamivudine (3TC, Epivir)
Tenofovir (TDF, Viread)
Emtricitabine (FTC, Emtriva)
Abacavir (ABC, Ziagen)
Stavudine** (D4T, Zerit)
Didanosine** (DDI, Videx)
Protease inhibitors
Lopinavir/ritonavir (LPV/r, Kaletra)
Atazanavir (ATV, Reyataz)
Fosamprenavir (fAMP, Lexiva)
Darunavir (DRV, Prezista)
Tipranavir* (TPV, Aptivus)
Saquinavir* (SQV, Invirase)
Nelfinavir* (NFV, Viracept)
Indinavir** (IDV, Crixivan)
Non-nucleoside reverse transcriptase inhibitors (NNRTIs)
Efavirenz (EFV, Sustiva)
Nevirapine (NVP, Viramune) Etravirine (ETV, Intelence)
Delavirdine** (DLV, Rescriptor)
Rilpivirine (Edurant)
Integrase inhibitors
Raltegravir (RAL, Isentress)
Elvitegravir
Dolutegravir (Tivacay)
Attachment inhibitor
Maraviroc (Selzentry)
Fusion inhibitor
Enfuvirtide* (T-20, Fuzeon)
P450 inhibitor (for “boosting” PIs)
Ritonavir (RTV or r, Norvir)
Cobicistat
*Uncommonly used. **No useful role

4. WHO 2013 recommendations for 1st-line ART in children < 3 y of age
“ABC should be considered the preferred NRTI whenever possible.”
LPV//r should not be given to infants < 14 d of age or in premature infants until after 14 d after their due date

5. WHO 2013: Start with LPV/r and switching to NNRTI?
NOTE:
The quoted study enrolled only children with history of SD NVP exposure
The quoted study used < 50 copies/ml as primary endpoint and < 1,000 c/ml as secondary endpoint- NOT < 400 c/ml as stated above.
The switch-to-NVP group had fewer cases of VL > 50 but more cases of VL > 1,000 and more resistance than group that stayed on LPV/r

6. WHO 2013 recommendations for 1st line ART in children > 3 y of age

7. Treatment failure: progressive steps, different definitions
Potency failure
Adherence failure
Virologic failure
Genotypic failure (resistance)
There are multiple definitions or types of treatment failure. If the regimen is inadequate or the dosage too low (potency failure), or if there is nonadherence (adherence failure), then virologic failure (a detectable viral load) will develop. Genotypic failure will develop, the time course depending on the genetic barrier to resistance and how high the viral load is- the lower barrier to resistance and higher the viral load, the more rapidly resistance is selected. With higher levels of viral load, eventually the CD4 stops going up and begins to fall (immunologic failure). Even later, the patient experiences renewed symptoms- clinical failure.
Immunologic failure
Clinical failure

8. Expected fall in viral load
Time
Log fold drop in viral load
Fold-drop in viral load
Viral load (example) --
600,000
100,000
1 month 2
100+
< 6,000
< 1,000
3 months 3
1,000
600
100
6 months 4
10,000 60
<50
If viral load at these follow up times is 3-fold or more than these examples, full suppression not likely

9. Days = 0 is day HAART started (6 weeks of age)
Days = 0 is day HAART started (6 weeks of age). Note initial rapid rise in viral load after infection at birth. Also not 2-phase decay in viral load on HAART- an initial rapid drop (about 1000-fold) in first 2-3 weeks, then slower decline to undetectable by ultrasensitive assay by 140 days of treatment. This girl is now 8 years old and has had an undetectable viral load since about 4 months of age.

10. Course of treatment failure
Start ZDV/3TC/NVP
Clinical deterioration
Gradual ZDV resistance
Nonadherence
NVP resistance
3TC resistance
CD4 Count
Viral load
The time course of treatment failure. The VL drops very rapidly after starting HAART, then more slowly. At the indicated point, the patient has a period of nonadherence. Very rapidly, NVP resistance (K103N mutation) is selected. What follows from this point will occur whether or not adherence improves. Within 1-3 months, 3TC resistance (M184V) is selected. This increases sensitivity to ZDV, but at this point ZDV is the only active drug- not enough to hold back the virus. The CD4 count has continued to improve, even after development of NVP resistance- we know that ZDV/3TC will make someone with AIDS better for a year or so. With time, however, the CD4 stops rising and starts to fall. One by one, ZDV-selected thymidine analogue mutations evolve. (TAMS: RT mutations at 41, 67, 69, 70, 210, 211, 215 and others- 215 and 41 being the most important.) Only as the viral load gets very high again and the CD4 has fallen does the patient have recurrent symptoms. At this point there is resistance to all 3 drugs with cross-resistance to EFV and complete or partial cross-resistance to all NRTIs.
1 year
2 years

11. Mechanism of reverse transcriptase inhibitors cDNA
NRTI = Nucleoside reverse transcriptase inhibitor
NNRTI = Non-nucleoside reverse transcriptase inhibitor
HIV reverse transcriptase
cDNA
ABC
HIV reverse transcriptase copies the viral RNA into DNA. The nucleoside analogues (D4T in this case), are added onto the growing DNA chain. But because they are blocked at one end, the next natural dNTP cannot be added- and the DNA chain is terminated. The NNRTIs work by binding to the RT molecule, blocking its activity.
NRTI (such as ABC(P3) is added onto cDNA chain, blocking further reverse transcription
NNRTI (NVP or EFV)
Blocks reverse transcriptase by binding at active site

12. The K103N mutation: how it can take over
Sensitive K103 HIV wild type (wt)
Resistant 103N HIV
NVP blocks sensitive HIV, not resistant HIV
Nevirapine
wt always present
NVP blocks the grown of the wild-type virus (with K at position 103). But the extremely rare, randomly produced mutant, with N at position 103, is able to grow. Within 2-4 weeks the mutant resistant virus takes over as the predominant virus in the blood.
In presence of NVP, only the resistant virus grows. Soon almost all virus is resistant!

13. Note that these patients were failing (by VL monitoring) for only 4-5 months- which is too early to detect failure using CD4 counts or clinical signs- yet they had developed significant resistance- nearly all NNRTI and 3TC resistance, and significant amount of other NRTI mutations. Note that these other mutations were more common in patients with VL > 4 log (> 10,000) copies/ml.
Drug resistance mutations in Thai patients failing D4T/3TC/NVP for an average of 4-5 months by viral load monitoring. Sungkanuparph S. CID 2007; 44:447–52

14. Nucleoside reverse transcriptase inhibitor resistance and cross-resistance
Zidovudine
(AZT)
Stavudine
(D4T)
Tenofovir
(TDF)
Abacavir
(ABC)
Didanosine
(DDI)
Lamivudine
(3TC) or Emtricitabine (FTC)
Thymidine analogue mutations
(TAMS)
Resistance
AZT
D4T
TDF
TAMS +184V
3TC, FTC > D4T, DDI, AZT, ABC > TDF
K65R
Resistance
TDF, ABC, DDI
D4T
3TC, FTC
Hypersensitivity
AZT
L74V
+ M184V
Resistance
ABC
DDI
3TC
FTC
Hypersensitivity
AZT
TDF
M184V
Resistance
3TC
FTC
Hypersensitivity
TDF > AZT
> D4T
Q151M
Resistance
AZT, D4T,
DDI, ABC >
TDF, 3TC, FTC

15. Summary: NRTI resistance patterns & options
Regimen
Common pattern
Other patterns
Geno
Resistance
Options*
D4T/3TC
M184V,
TAMS
3TC >
D4T, ZDV, ABC, DDI >TDF
TDF/3TC (+/-ZDV)>
(ZDV/3TC/ABC)
(how good depends on # of TAMS)
K65R, M184V or
M184V, Q151M
3TC, TDF, ABC, DDI, d4T
3TC, ZDV, D4T, DDI, ABC, (TDF)
ZDV/3TC
(TDF/3TC) “
TDF/3TC
K65R,
(M184V)
ABC/3TC
M184V, L74V
3TC, ABC, DDI
M184V, K65R
DDI/3TC
*Options: Underline = excellent, () = weak, others fair

16. NNRTI resistance 1st generation (nevirapine, efavirenz, delavirdine)
High potency but low genetic barrier to resistance
Most commonly K103N- resistance to all 1st generation
NVP also selects for Y181C (especially in newborns) which has mild effect on EFV but associated with increased failures
Other mutation patterns also seen
2nd generation (Etravirine)
NO EFFECT of K103N
Resistance increases with other NNRTI mutations- 3 or more yield

18. Protease inhibitor resistance
Some PIs select for drug-specific mutations (e.g. NFV, ATV)
Some PIs can be boosted or unboosted (ATV, fAPV)- low-level resistance may be clinically significant if not boosted
Boosted PIs more durable
Resistance to LPV requires 5-10 mutations
Virologic failure while receiving LPV/r usually due to nonadherence
Prolonged virologic failure while on LPV/r eventually will lead to LPV resistance

19. Drug resistance testing
Commercial methods start with RT PCR of bulk virus in plasma
Not sensitive to minor strains- e.g. genotyping (sequencing) cannot detect strain representing < 10-25% of circulating virus
When to get resistance testing (resource-rich)
Baseline: resistant strains (especially NNRTI resistance) circulating in population
Whenever resistance suspected (e.g. failing and patient appears adherent)
Selective pressure- patient taking medication

20. HIV drug resistance genotype
RT PCR bulk plasma virus to produce cDNA
Sequence pol gene
Derive predicted amino acid sequence
Identify mutations known to confer resistance (e.g. Stanford database, IAS-USA, etc.)
Virtual phenotype™ genotype interpretation
Identified set of significant mutations is matched with massive database of genotype-phenotype correlations
Reported as predicted fold resistance (used to also give number of matches)
Fold-resistance interpreted according to in vitro or clinical measure of activity

22. IAS-USA, Topics in HIV Medicine, March 2013

23. IAS-USA, Topics in HIV Medicine, March 2013

26. Interpretation of resistance testing
Complete treatment and viral load history are essential
What is current regimen and is patient taking it?
Many resistance mutations cause decreased viral fitness: if there is not selective pressure, wild-type virus rapidly outgrows mutant, but archive of mutant remains

27. WHO 2013

28. Johnny B. Goode Newly infected adolescent CD4 = 320, VL = 58,000
Prescribed TDF/FTC/ATV/r
VL 1 month later = 5,200
VL at 2 months = 4,000
Genotype is wild-type
How do you interpret this situation?
How to proceed?