1. Antiretroviral Drug Resistance
Anna Maria Geretti

2. Virus-related factors Viral load Drug susceptibility Fitness
Host-related factors
Adherence
Tolerability
Immunity
Genetics
Drug-related factors
Potency
PK properties
Genetic barrier
Virus-related factors
Viral load
Drug susceptibility
Fitness
Drug pressure
Persistent virus replication during HAART
Emergence and evolution of drug resistance 2 2

3. Attachment Fusion Release of RNA
Assembly
Reverse transcription Integration Transcription
Maturation and budding

4. Characteristics of HIV-1 infection
High virus replication rate ( virus particles / day)
Rapid virus clearance (T½ cells producing virus: <1 day; T½ free virus in plasma: a few hrs)
Virus latency (1:106 resting CD4 T cells)
Chronic immune activation
CD4 T-cell depletion ( cells lost daily)
Progressive immune deficient state
Continuous virus genetic evolution
Wong 1997; Chun 1997; Siliciano 2003; Strain 2003; Han 2007 4

5. Mechanisms of HIV genetic evolution RT-driven mutagenesis
Rate: ~1 wrong incorporation per genome round
All possible point mutations generated daily
APOBEC-driven mutagenesis
Deamination of cytosine residues in nascent DNA
GA hypermutation
Recombination
Rate: 7-30 per genome round
Hybrid virus progeny produced from different strains
Guanosine to adenosine

6. Dominant quasispecies
Escape from pressure
Preserved fitness
Within the quasispecies, positive (Darwinian) selection implies that one or more members of the quasispecies are better suited to a given environment while negative selection eliminates unfit variants
In the absence of drug pressure, resistant mutants tend to disappear from the dominant quasispecies as they are outgrown by the fitter wild-type virus. The greater the impact on fitness the more rapid the disappearance
rapid turnover
rapid adaptation

7. Consequences of HIV genetic variability
At the population level
Continuous emergence of new variants
At the patient level
Escape from immune pressure
Escape from drug pressure
Increased fitness and pathogenicity
Challenge for diagnostic and monitoring assays

8. Emergence and evolution of resistance
Single mutant Double mutant Triple mutant
Thus, resistant variants pre-exist the introduction of drug pressure. Once drug pressure is introduced, if replication continues the resistant mutant will acquire a selective advantage and gradually emerge as dominant
Evolution subsequently occurs so that multiple mutations come to coexist on the same genome
Resistance mutations occur in regions of the viral genome that play a key role in virus replication and infectivity. As a result they come at a cost of reduced viral fitness, which can be described as the virus ability to replicate and infect in a given environment. Over time however, the virus tends to acquire further changes that compensate for the reduced fitness and restore virus replication at least partially. Thus we can say that the natural evolution of HIV under drug pressure is towards increasing resistance and preserved fitness
Increasing number of mutations
Accumulation of mutations on the same viral genome
Initially reduced viral fitness
Compensatory changes restore fitness

9. Key principles of resistance
Drug-resistant mutants are selected (not created) by drug pressure if virological suppression is incomplete
Ongoing virus replication under drug pressure leads to the evolution of resistance and cross-resistance
Resistant mutants often display reduced fitness but compensatory changes emerge over time that partially restore virus fitness
We can summarise these observations as follows

10. PCR Viral gene (e.g., RT) HIV RNA Plasma Sequencing Mutations RT M184V
Methionine  Valine
@ codon 184 of RT
ATG / AUG  GTG / GUG
This slides illustrates how genotypic resistance test is performed in the laboratory

11. escalating drug concentrations
PCR
Viral gene (e.g., RT)
HIV RNA
Plasma
Culture with
escalating drug concentrations
Fold-changes in IC50 relative to wild-type
M184V = 100 FC for 3TC
Defective laboratory HIV vector (e.g., RT_)
Infectious HIV

12. Detection of resistant mutants
0.001
0.01
0.1 1 10
100
Detected by ultrasensitive
methods
Mutation Frequency
Detected by routine methods
Natural background
With this standard resistance testing method we can detected the most prevalent mutants. In research settings we can use more sensitive techniques (e.g., real-time PCR) to detect mutants present at levels between 10-20% and ~0.1%. Clinically it is not considered useful to detect rarer mutants (<0.1%) as these will be part of the “natural background” to be expected in all patients regardless of drug pressure
We have increasing evidence that the low-frequency mutants present at levels between 10-20% and 0.1% matter, but no way of detecting them in routine diagnostic settings. Thus, expert and integrated interpretation of treatment history and resistance results is paramount.

13. Low-frequency resistance in the FIRST study
Mutations
Resistance test P
Standard
UDS
NNRTI 7%
15%
<0.001
NRTI 6%
14% PI 2% 5%
0.03
Any
28%
N=258
Background.Minor (i.e., <20% prevalence) drug‐resistant human immunodeficiency virus (HIV) variants may go undetected, yet be clinically important.
Objectives.To compare the prevalence of drug‐resistant variants detected with standard and ultra‐deep sequencing (detection down to 1% prevalence) and to determine the impact of minor resistant variants on virologic failure (VF).
Methods.The Flexible Initial Retrovirus Suppressive Therapies (FIRST) Study (N = 1397) compared 3 initial antiretroviral therapy (ART) strategies. A random subset (n = 491) had baseline testing for drug‐resistance mutations performed by use of standard sequencing methods. Ultra‐deep sequencing was performed on samples that had sufficient viral content (N= 264). Proportional hazards models were used to compare rates of VF for those who did and did not have mutations identified.
Results.Mutations were detected by standard and ultra‐deep sequencing (in 14% and 28% of participants, respectively;  ). Among individuals who initiated treatment with an ART regimen that combined nucleoside and nonnucleoside reverse‐transcriptase inhibitors (hereafter, “NNRTI strategy”), all individuals who had an NNRTI‐resistance mutation identified by ultra‐deep sequencing experienced VF. When these individuals were compared with individuals who initiated treatment with the NNRTI strategy but who had no NNRTI‐resistance mutations, the risk of VF was higher for those who had an NNRTI‐resistance mutation detected by both methods (hazard ratio [HR], [95% confidence interval {CI}, 3.41–45.10]) and those who had mutation(s) detected only with ultra‐deep sequencing (HR, 2.50 [95% CI, 1.17–5.36]).
Conclusions.Ultra‐deep sequencing identified a significantly larger proportion of HIV‐infected, treatment‐naive persons as harboring drug‐resistant viral variants. Among participants who initiated treatment with the NNRTI strategy, the risk of VF was significantly greater for participants who had low‐ and high‐prevalence NNRTI‐resistant variants.
Risk of failure of first-line NNRTI-based ART in patients with NNRTI resistance
Bulk resistance: HR 12.4 [ ]
UDS resistance: HR [ ]
USD = Ultra deep sequencing
Siemen, JID 2009 13 13

14. Drug pressure
Resistant
Wild-type
Limit of detection
20-30%

15. Key principles of resistance
Once drug pressure is removed, resistant mutants are outgrown by fitter wild-type virus and become undetectable by routine tests
Resistance test results obtained after therapy is discontinued are not reliable
Resistant mutants persist at low frequency in plasma and are “archived” in latently infected cells
Resistance is long-lasting
Resistance test results must be interpreted in the context of the patient’s treatment history
The clinical implications of these observations can be summarised as follows 15

16. VL Ms S., 35 yr Δ HIV+ Dec 1997 M184V = 3TC, FTC Y181C = NNRTIs
Wild-type
Resistant
M184V = 3TC, FTC
Y181C = NNRTIs
d4T 3TC
NVP
d4T 3TC NVP VL
<50
M184V
Y181C
Testing should be undertaken on samples taken while the patient is receiving therapy

17. Transmitted drug resistance
Stable after transmission
Gradual reversion over time, sometimes incomplete
Persistence at low frequency in plasma
Persistence in latently infected cells
Drug pressure
Transmission
Finally, a few considerations about transmitted drug resistance. We have discussed the dynamics of resistant mutants during treatment and seen that mutants selected during therapy disappear rapidly once therapy is interrupted
Transmitted resistant mutants however behave differently and can persist for long periods in the absence of drug pressure. This cartoon explain the proposed model. HIV transmission leads to an infection which is initially highly homogenous. If the virus transmitted is resistant the infection will become established with a homogenously resistant virus. There is no wild-type virus that can rapidly out compete the resistant mutant. For the wild-type to occur the resistant mutants is required to back mutate, a process that is rather slow due to the fact that the transmitted virus is genetically constrained
Reversion does occur gradually over time, but the resistant mutants will persist as minority species and archived resistance, with the potential for a long-lasting impact

18. Genetic barrier Residual activity Hypersusceptibility
Key concepts
Genetic barrier Residual activity Hypersusceptibility

19. Genetic barrier to resistance
Defined by:
Number of mutations required to compromise activity
Impact of each mutation on drug susceptibility
Interactions between mutations
Fitness cost of resistance
Drug concentration
Resistant
Wild-type 19

20. Genetic barrier – A simplified overview
Class
ARVs
Genetic barrier
NRTIs
ZDV/3TC, d4T/3TC ++
ABC/3TC, TDF/3TC +
TDF/FTC
NNRTIs
EFV, NVP, ETV, RPV
ETV, (RPV)
+/++
PIs
Boosted
+++/++++
Fusion inhibitors
T20
CCR5 antagonists
MVC
++ (for R5 virus)
Integrase inhibitors
RAL, ELV 20

21. Common NRTI resistance patterns
NRTIs
Mutations
ZDV
d4T
ABC
ddI
TDF
3TC
FTC
ZDV 3TC
d4T 3TC
M184V
TAMs
+ TAMs
d4T 3TC TDF 3TC TDF FTC
K65R
ABC 3TC
L74V
Y115F
TAMs = thymidine analogue mutations: M41L, D67N, K70R, L210W, T215Y/F, L219Q/E

22. Resistance with first-line HAART
Study
ART wk
Tests
K65R
L74V
M184V
TAMs
3rd drug
GS903 n=299
TDF 3TC EFV 48 29
24%
41%
EFVR 55%
GS934 n=244
TDF FTC EFV 96 14
14%
EFVR 71%
GS934 n=243
ZDV 3TC EFV
31% 3%
EFVR 62%
CNA n=770
ABC 3TC EFV 38
21%
47%
EFVR 58%
ABT418 n=190
TDF FTC LPV/r 23
17%
PIR 0
SOLO n=190
ABC 3TC FPV/r 32
12%
ARTEMIS n=343
TDF FTC DRV/r 31 6%
STARTMRK n=281
TDF FTC RAL 13
38%
RALR 33%
ITT analysis
RAL arm: Integrase from 12/39 VFs = 4 RALR*; RT in 13/39 VFs = 5 FTCR
EFV arm: RT from 9/45 VFs = 5 EFVR; 2 FTCR
Margot, HIV Med 2006; Margot, JAIDS 2009; Moyle, JAIDS 2005; Molina, IAC 2004; Gathe, AIDS 2004; Mills, AIDS 2009; Lennox, Lamcet 2009.

23. 1st and 2nd generation NNRTIs
Nevirapine
Efavirenz
Etravirine
Major resistance mutations
L100I, K101E/P
K103N/S
V106A/M
E138K, V179F
Y181C/I/V, Y188L/H/C, G190A/S/E
F227C, M230L, K238T
NNRTI resistance mutations emerge rapidly in patients with detectable viraemia during NNRTI therapy
Single NNRTI mutations can cause high-level NNRTI resistance and cross-resistance
Resistance mutations continue to accumulate
Rilpivirine 23

24. Patients with VL <50 copies/ml at wk 48 (ITT-TLOVR)
Activity of ETV with a strong backbone
DUET studies: OBR (with DRV/r) + ETV or Placebo
Patients with VL <50 copies/ml at wk 48 (ITT-TLOVR)
100 90 80 70 60 50 40 30 20 10
ETV + OBR (n=599)
Placebo + OBR (n=604)
61%
Responders (%) ± 95% CI
40%
p<0.0001*
Time (weeks)
ART-experienced patients with documented NNRTI and PI resistance
Katlama, AIDS 2009

25. Activity of ETV with a weak backbone Study TMC125-C227: 2 NRTIs + ETV or PI
Viral rebound was most apparent in individuals who had more than one NRTI mutation at baseline. In contrast, the subset of patients with no NRTI mutations demonstrated a more durable viral load suppression of about 1.75 log10 copies/mL below baseline as of week 16. Those patients with four or more NRTI mutations at baseline showed virtually no response to etravirine. The number of NNRTI mutations also had an impact on etravirine efficacy, and there was transient suppression in some individuals who had just one NNRTI mutation at baseline.
ART-experienced, PI-naive patients with documented NNRTI resistance
Ruxrungtham, HIV Med 2008

26. The genetic barrier of PIs in vitro
450
DRV (R41T, K70E)
RTV (G16E, M46I, V82F, I84V)
SQV (G48V, A71V, G73S, I84V, L90M)
NFV (L10F, D30N, R41K, K45I, M46I, V77I, I84V, N88D)
APV (L10F, V32I, L33F, M46I, I47V, I50V)
LPV (L10F, L23I, M46I, I50V, I54V, L63P, V82A)
TPV (L33V, M46L, V82T)
ATV (L10F, V32I, M46I, I62V, A71V, I84V, N88S)
400
350
300
Increase in PI selection concentration
250
200
150
100 50
100
300
500
700
900
1100
Time (days)
De Meyer, Antimicrob Agents Chemother 2005;
De Meyer, IHDRW 2006

27. VFs with developing mutations (%)
Emergence of PI mutation with DRV/r vs LPV/r TITAN study: OBR + DRV/r or LPV/r
TITAN 96 week analysis
p0.05*
p0.05*
DRV/r
LPV/r 40
24/72 (33%)
19/72 (26%) 30
6/39 (15%)
VFs with developing mutations (%) 20
3/39 (8%) 10
Primary PI mutations1
NRTI RAMs1
*Exact Chi-Squared Test; TITAN 96 week analysis
1Johnson et al. Top HIV Med 2007;
ART-experienced, LPV- and DRV-naive patients
De Meyer, HIV 27

28. Resistance as a continuum
Lower cut-off = Level of resistance beyond which response begins to fall off
Upper cut-off =
Level of resistance beyond which
clinical response is lost
Zone of
intermediate
response
Response
Resistance

29. How to calculate the GSS
Genotypic Susceptibility Score
RT mutations: M41L T215Y K103N
Protease mutations: None
Regimen: TDF TC LPV/r
Resistance: Intermediate Susceptible Susceptible
Score:
GSS ?

30. Change in plasma viral load
Partial treatment interruption in patients with resistance reveals residual activity
NRTI PI
NNRTI
T20
–0.5
0.0
0.5
1.0
Discontinued treatment class
Change in plasma viral load
Week 2 change in VL
Deeks, CROI 2005

31. Mechanisms of NRTI resistance: Primer unblocking
T215Y-mediated resistance
Hydrolytic removal of the chain-terminating NRTI enables DNA synthesis to resume
The pyrophosphate donor in most cells is ATP
M184V antagonizes the process
3TC partially restores susceptibility to ZDV, d4T and TDF in the presence of TAMs
3TC antagonizes the emergence of TAMs P
Pyrophosphate can also be an acceptor
Antagonised by M184V, K65R, L74V
Gotte, J Virol 2000

32. Key principles of resistance
Resistance moves along a continuum and increasing numbers of mutations lead to progressive loss of responses
Residual activity is possible despite the presence of extensive resistance (best evidence for the NRTIs)
Resistance carries a fitness cost that reduces viral replication
Antagonistic effects between mutations can have beneficial effects
The clinical implications of these observations can be summarised as follows

33. Clinical implications for patients with treatment failure
The likelihood of drug-resistance depends upon the drug, the regimen and the level of adherence
When selecting a new regimen, aim for a GSS ≥2
Avoid functional monotherapy with drugs that have a low genetic barrier
If options are limited, exploit residual activity and hypersusceptibility effects – continue the NRTIs rather than stopping therapy
The clinical implications of these observations can be summarised as follows 33

34. Advanced Virology Workshop
Are you curious about what to do when a patient suddenly stops NNRTI-based ART?
What “undetectable” viral load really means?
How we can easily predict HIV-1 tropism?
Advanced Virology Workshop
Ongoing
Thank you 34