HIV Drug Resistance Training
Module 6:
Sequencing Interpretation
and Analysis
1
Topics




Understanding Mutations
Identifying Resistant Mutations
Phylogenetic Analysis
Subtype Determination
2
Objectives
Identify information needed by physicians and
policy makers to make treatment decisions on a
population basis.
 Describe algorithms used to translate sequence
results to drug susceptibility predictions.
 Analyze results to determine HIV subtypes.
 Identify how to complete phylogenetic analysis.

3
understanding mutations
What are the different types of mutations?
How are mutations identified?
4
Genotype Interpretation: The
Challenge
K65R
K70R
E44D L74V
M41L
V118I V75M T215Y
K219N
Q151M T215F
M184V K103N T69SSA
F227L
K219E
K101P
G190A
V106M
“Sensitive” to ??
“Resistant” to ??
“Maybe” to ??
I50V
M46I
G48V
A71V
V32I
V82A
I54V
I47V
L90M
D30N
V82F
N88S
I84V
5
Mutation


Molecular definition: change in nucleic acid sequence
compared to a reference sequence
Biological definition: change in nucleic acid sequence that
results in a change in structure or function of the nucleic
acid or a resulting protein
Codon
AAA GAC AGT
AAA AAC AGT
Silent Mutation
AAA GAC AGT
AAA AAC AGC
Lys Asp Ser
(K) (D) (S)
Lys Asn Ser
(K) (N) (S)
6
Mutational Nomenclature
G48V
Wild-type (wt) amino acid
(consensus or reference)
Mutant amino acid
Codon position
PR: 1-99 amino acids
RT: 1-540 amino acids
L10L/I (mix of wt and mutant)
V82A/F (mix of 2 mutants)
7
Effect of Nucleotide Changes
Nucleotide changes (mutations)

Changes in amino acid sequence of a protein

Changes in structure/function of the protein (e.g. PR or RT)

Changes in ability of drug to inhibit target enzyme (resistance)
8
Types of Mutations

Polymorphisms
– Naturally occurring mutations, not selected by drugs
(but can influence susceptibility)

“Primary” mutations
– Directly affect drug binding, present near active site
– Appear first in pathway to resistance
– Not present in virus not exposed to drug pressure

“Secondary” mutations
– Compensate for fitness defects
– Do not usually confer resistance on their own but
modulate susceptibility
– May include polymorphisms that are found more
frequently in resistant viruses
9
Residues Associated with HIV PI Resistance
Primary 48, 50, 82, 84…
Flap 46, 47, 53, 54…
Secondary 10, 20, 32,
36, 63, 71…
How are Drug Resistance Mutations
Identified?




Researchers identify mutations selected by the drug
during in vitro selection, phase I studies, site-directed
mutagenesis
BUT…
Drug resistance mutations identified during drug
development (esp. in vitro) may not be the most
relevant mutations in clinical settings.
Mutations that are sufficient to cause drug resistance
may not be necessary to effect drug resistance.
There may be cross-resistance due to mutations
selected by related drugs.
11
Discussion


What are the different types of mutations?
How are mutations identified?
12
identifying resistance mutations
How can you identify which mutations are responsible for drug
resistance in your population?
13
Basis for Drug Resistance Knowledge
Genotypic-phenotypic correlations on laboratory
isolates (often confirmed by site-directed
mutagenesis studies)
2. Genotypic-phenotypic correlations on clinical
HIV-1 isolates
3. Correlations between HIV-1 genotype and the
treatment history of patients from whom
sequenced virus isolates are obtained
4. Correlations between HIV-1 genotype and the
virologic response to a new treatment regimen.
1.
14
http://hivdb.stanford.edu/pages/documentPage/drm.html
Selected Resistance and Cross-Resistance
Exposure to new drug X
Mutations A, B, C, D, E, F
Exposure to other drugs
from same class
Mutations A, D, E, G, H, I
Exposure to drugs from
other classes
Mutations J, K, L…
Some mutations contribute to resistance (decrease susceptibility)
Others may suppress resistance (increase susceptibility)
15
Sources of Genotype Interpretation
Information








IAS-USA (Hirsch et al., JAMA 2000; annual+ updates)
– Expert consensus; updated frequently
Stanford (R. Shafer), www.HIVResistance.com
– Comprehensive, updated frequently, good notes
Resistance Collaborative Group (DeGruttola et al., 2000)
– Initially used in GeneSeq assay, not currently used
GART (Baxter et al., 2000), VIRADAPT (Durant et al., 1999)
– used in prospective clinical trials
TruGene, ViroSeq, other test providers
ABL/Virology Networks
European Resistance guidelines
ANRS, other EU country-specific guidelines
16
International AIDS SocietyUSA* Drug
Resistance Mutations Group

IAS-USA information available at:
http://www.iasusa.org/resistance_mutations/index.html.
*The International AIDS Society–USA (IAS–USA) is a not-for-profit, HIV clinical specialisteducation organization. It is entirely different from and not affiliated with the International AIDS
17
Society (Stockholm, Sweden).
Multi-NRTI Resistance Mutations (IAS-USA)
18
Johnson et al. Topics HIV Med. 16(5): 138, December 2008. Updated on www.iasusa.org.
© 2008. The International AIDS Society–USA
Individual NRTI Resistance Mutations: IAS-USA
19
Johnson et al. Topics HIV Med. 16(5): 138, December 2008. Updated on www.iasusa.org.
© 2008. The International AIDS Society–USA
NNRTI Resistance Mutations (IAS-USA)
20
Johnson et al. Topics HIV Med. 16(5): 138, December 2008. Updated on www.iasusa.org.
© 2008. The International AIDS Society–USA
PI Resistance Mutations (IAS-USA)
21
Johnson et al. Topics HIV Med. 16(5): 138, December 2008. Updated on www.iasusa.org.
© 2008. The International AIDS Society–USA
NRTI Resistance Mutations (Stanford)
Mutations in bold red are associated with higher levels of phenotypic resistance or
clinical evidence for reduced virological response.
22
http://hivdb.stanford.edu/cgi-bin/NRTIResiNote.cgi
NNRTI Resistance Mutations (Stanford)
Mutations in bold red are associated with higher levels of phenotypic resistance or
clinical evidence for reduced virological response.
23
http://hivdb.stanford.edu/cgi-bin/NNRTIResiNote.cgi
PI Resistance Mutations (Stanford)
Mutations in bold have been shown to reduce in vitro susceptibility or in vivo
virological response. Mutations in bold underline are relative contraindications to
the use of specific PIs.
24
http://hivdb.stanford.edu/cgi-bin/PIResiNote.cgi
Mutations vs. Rules

Mutations: amino acid changes in a patient
sequence relative to some reference (e.g. HXB2,
NL4-3, or consensus B)
– Some mutations are associated with drug resistance

Rules: specify certain mutations or combinations
of specific mutations as being determinants of
drug resistance
– e.g. “T215Y plus 2 or more TAMs”

Simple lists of mutations (e.g. IAS-USA,
HIVResistanceWeb) are NOT rules!
25
Stanford HIVdb Interpretation (1 of 2)
26
Stanford HIVdb Interpretation (2 of 2)
27
Discussion

How can you identify which mutations are
responsible for drug resistance in your
population?
28
phylogenetic analysis
What is phylogenetic analysis, and why is it useful?
29
Phylogenetic analysis



QA (detection of contamination)
Identify epidemiological linkages
Study mutation pathways
30
Phylogenetic Analysis

Compare a test sequence to others
– Tested in same batch or over recent period of time
– Collected at the same site
– Related sequences with possible index-source
relationship
Phylogenetic relationships can be described
quantitatively and use statistical methods to test
significance of linkages
 Challenging to decide on absolute thresholds to
define 2 viruses as being "highly related" etc.
(98%? 99%?)

31
Phylogenetic Tree
Clusters of highly related sequences
Reference sequences
32
100
7028
307l
100
100
C7018
5009
5043
5017
7008
T3046
E5025
3044
02_MP1211
84
02_DJ.DJ263
100
3031NV
T3003
100
02_NG.IBNG
01_TH.CM240
98
01_TH.93TH253
01_CF.90CF402
84
A3_DDI579
A3_DDJ360
100
100
90
A3_DDJ369
A1_SE.SOSE7253
96
A1_UG.92UG037
A1_KE.Q2317
E5047
E5056
7085
100
7022
5011
100
G_BE.DRCBL
G_SE.SE6165
G_FI.HH8793
06_95ML84
100
06_BFP90
06_97SE1078
100
K_97CD.EQTB11
K_96CM.MP535
F2_CM53657
100
F2_96CM.MP257
F2_96CM.MP255
100
05_X492
05_VI961
F1_BE.VI850
100
F1_FI.FIN9363
F1_BR.93BR020
78
D_UG.94UG114
D_99TCMN011
D_ZR.NDK
100
B_US.WEAU160
B_US.JRFL
B_FR.HXB2R
CRF02
CRF01
A3
A1
G
CRF06
K
F2
CRF05
F1
0.02
D
B
subtype determination
What are subtypes and how are they determined?
34
HIV-1 Subtypes
gag
pol
env
35
Global Distribution of HIV-1 Genetic
Subtypes (2004)
36
Hemelaar et al. AIDS 2006, 20:W13–W23
Subtype Determination

Simplistic Approach
– Compare test sequence to set of reference sequences
– Subtype of closest matching reference sequence is
assigned to test sequence

Reality: recombination between subtypes
– PR matches one subtype; RT matches another
– Circulating recombinant forms: found in unlinked
individuals
– Unique recombinant forms (URF)
– Look for similarity to reference sequences using
substrings of test sequence; "sliding window"
37
Circulating Recombinant Forms (CRF)
CRF02_AG
CRF06_cpx
38
http://www.hiv.lanl.gov/content/sequence/HIV/CRFs/CRFs.html
Naturally Occurring Amino Acids in Non-B
Subtype HIV-1
Position
98
179
181
190
10
20
36
46
69
71
93
NNRTI
Amino Acid
Subtypes
G*
C
D*, I
nd
C
Group O
A*
C
PI
I, V
A, F, J, group O
R, M, I
Most non-B
I, L
Most non-B, group O
I*
C, F, AE
K
Most non-B
V
Group O
L
C
* isolated
observations
nd = not defined
39
Adapted from Parkin and Schapiro, Antiviral Therapy 9: 3-12, 2004
Subtype-Specific PR Polymorphisms
(Untreated Patients)
40
Kantor R et al. PLoS Medicine 2005 Apr;2:112
Subtype-Specific Treatment (PI)-Related
Mutations
41
Kantor R et al. PLoS Medicine 2005 Apr;2:112
Are Subtype B Resistance Mutations also
Treatment-related in Non-Bs?
Each of the 55 known DR mutations occurred in at
least one non-B isolate; 44 (80%) of these
mutations were significantly associated with
therapy in non-B isolates
 Most positions associated with DR in subtype B
viruses are selected by antiretroviral therapy in
one or more non-B subtypes as well.
 No evidence that non-B viruses develop mutations
at positions that are not associated with resistance
in subtype B viruses.
 Based on currently available data, global
surveillance efforts and genotypic assessments of
DR should focus primarily on the known subtype B
42
drug-resistance mutations.

Kantor R et al. PLoS Medicine 2005 Apr;2:112
HIV-1 Subtype Influences Mutation
Preference
Drug
NFV
EFV
d4T
Subtype C
L90M
V106M
K65R
Subtype B
D30N
K103N
TAMs
Different subtypes may develop different profiles
of mutations in response to the same treatment
 However, once detected, the interpretation of
the meaning of these mutations is independent
of subtype

43
Subtype Determination
Subtype is not a critical factor for genotype
interpretation
 Different prevalence of polymorphisms and
secondary mutations
 Different mutational pathway preferences
 Geographic and/or epidemiological clustering

44
What about HIV-2?
Naturally Occurring Amino Acids in SIV and HIV-2 Implicated in Modulation of
Antiretroviral Drug Susceptibility (at Positions Selection form HIV-1 Knowledge Base)
NRTI
NNRTI
PI
FI (gp41)
Position
Amino
acid
Position
Amino
acid
Position
Amino
acid
Position
Amino
acid
69
N
101
A, E, P
10
V
42
Q
75
I
103
C, M, R
20
V
43
Q
118
I
106
I
32
I
210
N
179
T
36
I
215
S
181
I, V
46
I, V
219
E
188
F, L, W
47
V
190
A
71
I, V
227
Y
77
T
82
I
Drugs impacted: NNRTI (all), PI (APV, ATV, TPV, DRV?)
45
Parkin and Schapiro, Antiviral Therapy 9: 3-12, 2004
Discussion

What are subtypes and how are they
determined?
46
Discussion
What information do physicians and policy
makers need to make treatment decisions on a
population basis?
 How can we get them this information?
 What sources of information/interpretation would
be useful to us?

47
Small Group Exercise
Work in small groups.
Read the directions
Look through some sample sequences (five
pages).
 Identify mixtures.



48
Example 1
49
Example 2
50
Example 3
51
Example 4
52
Example 5
53
Reflection
What information do physicians and policy
makers need to make treatment decisions on a
population basis?
 How can we get them this information?
 What sources of information/interpretation would
be useful to us?

54
Summary




Understanding Mutations
Identifying Resistant Mutations
Subtype Determination
Phylogenetic Analysis
55