HIV Drug Resistance Training Module 6: Sequencing Interpretation and Analysis Topics     Understanding Mutations Identifying Resistant Mutations Phylogenetic Analysis Subtype Determination.

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Transcript HIV Drug Resistance Training Module 6: Sequencing Interpretation and Analysis Topics     Understanding Mutations Identifying Resistant Mutations Phylogenetic Analysis Subtype Determination.

HIV Drug Resistance Training

Module 6: Sequencing Interpretation and Analysis 1

Topics

    Understanding Mutations Identifying Resistant Mutations Phylogenetic Analysis Subtype Determination

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.

understanding mutations

What are the different types of mutations?

How are mutations identified?

Genotype Interpretation: The Challenge

M41L K65R V118I K70R E44D V75M L74V T215Y K219N Q151M M184V K219E V106M T215F K103N F227L G190A T69SSA K101P D30N I50V M46I V32I V82A A71V I47V I54V G48V L90M V82F N88S I84V

“Sensitive” “Resistant” to ??

to ??

“Maybe” to ??

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 A AC AGT Silent Mutation AAA GAC AGT AAA A AC AG C Lys (K) Asp (D) Ser (S) Lys (K) Asn (N) Ser (S)

Mutational Nomenclature

G48V Wild-type (wt) amino acid (consensus or reference) Codon position PR: 1-99 amino acids RT: 1-540 amino acids Mutant amino acid

L10L/I (mix of wt and mutant) V82A/F (mix of 2 mutants)

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)

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

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 mutagenesis selection, phase I studies, site-directed 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.

Discussion

  What are the different types of mutations?

How are mutations identified?

identifying resistance mutations

How can you identify which mutations are responsible for drug resistance in your population?

Basis for Drug Resistance Knowledge

Genotypic-phenotypic correlations on laboratory isolates (often confirmed by site-directed mutagenesis studies) Genotypic-phenotypic correlations on clinical HIV-1 isolates Correlations between HIV-1 genotype and the treatment history of patients from whom sequenced virus isolates are obtained Correlations between HIV-1 genotype and the virologic response to a new treatment regimen.

http://hivdb.stanford.edu/pages/documentPage/drm.html

Selected Resistance and Cross-Resistance

Exposure to new drug X Exposure to other drugs from same class Exposure to drugs from other classes Mutations A, Mutations A, Mutations J, B, C, D, D, K, E, L… E, F G, H, I

Some mutations contribute to resistance (decrease susceptibility) Others may suppress resistance (increase susceptibility)

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

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 specialist education organization. It is entirely different from and not affiliated with the International AIDS Society (Stockholm, Sweden).

Multi-NRTI Resistance Mutations (IAS-USA)

Individual NRTI Resistance Mutations: IAS-USA

NNRTI Resistance Mutations (IAS-USA)

PI Resistance Mutations (IAS-USA)

NRTI Resistance Mutations (Stanford)

Mutations in bold red are associated with higher levels of phenotypic resistance or clinical evidence for reduced virological response.

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.

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.

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!

Stanford HIVdb Interpretation (1 of 2)

Stanford HIVdb Interpretation (2 of 2)

Discussion

 How can you identify which mutations are responsible for drug resistance in your population?

phylogenetic analysis

What is phylogenetic analysis, and why is it useful?

Phylogenetic analysis

   QA (detection of contamination) Identify epidemiological linkages Study mutation pathways

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%?)

Phylogenetic Tree

Clusters of highly related sequences Reference sequences

90 78 100 100 84 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 84 96 98 100 C7018 100 7028 307l 5017 5009 5043 E5025 3044 7008 T3046 02_MP1211 02_DJ.DJ263

02_NG.IBNG

E5056 7085 E5047 7022 5011 06_BFP90 D_ZR.NDK

U_83ZR003 3031NV T3003 01_TH.CM240

06_95ML84 06_97SE1078 K_96CM.MP535

05_X492 U_90CD121E12 H_BE.VI997

01_TH.93TH253

01_CF.90CF402

A3_DDI579 A3_DDJ360 A3_DDJ369 A1_SE.SOSE7253

G_SE.SE6165

F2_CM53657 05_VI961 B_US.JRFL

B_FR.HXB2R

A1_UG.92UG037

A1_KE.Q2317

G_BE.DRCBL

G_FI.HH8793

K_97CD.EQTB11

F2_96CM.MP257

F2_96CM.MP255

F1_BE.VI850

F1_FI.FIN9363

F1_BR.93BR020

D_UG.94UG114

D_99TCMN011 B_US.WEAU160

H_BE.VI991

H_CF.90CF056

C_98SN.86HPD

C_BW.96BW0502

C_IN.21068

C_ET.ETH2220

J_97CD.KTB147

J_SE.SE92809

J_SE.SE91733

CPZ_GAB CRF06

subtype determination

What are subtypes and how are they determined?

gag

HIV-1 Subtypes

pol env

Global Distribution of HIV-1 Genetic Subtypes (2004)

Hemelaar et al. AIDS 2006, 20:W13 –W23

Subtype Determination

  Simplistic Approach – Compare test sequence to set of reference sequences – Reality: recombination between subtypes – PR matches one subtype; RT matches another – – – Subtype of closest matching reference sequence is assigned to test sequence Circulating recombinant forms: found in unlinked individuals Unique recombinant forms (URF) Look for similarity to reference sequences using substrings of test sequence; "sliding window"

Circulating Recombinant Forms (CRF)

CRF02_AG CRF06_cpx

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 Amino Acid NNRTI G* D*, I C A* PI I, V R, M, I I, L I* K V L Subtypes C nd Group O C A, F, J, group O Most non-B Most non-B , group O C, F, AE Most non-B Group O C * isolated observations nd = not defined

Adapted from Parkin and Schapiro, Antiviral Therapy 9: 3-12, 2004

Subtype-Specific PR Polymorphisms (Untreated Patients)

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 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

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

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) Position 69 75 118 210 215 219

NRTI

Amino acid N I I N S E 101 103 106 179 181 188 190 227

NNRTI

Position Amino acid A, E, P C, M, R I T I, V F, L, W A Y Position 10 20 32 36 46 47 71 77 82

Amino acid V V I I I, V V I, V T I

FI (gp41)

Position 42 43 Amino acid Q Q

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?

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?

Small Group Exercise

    Work in small groups. Read the directions Look through some sample sequences (five pages).

Identify mixtures.

Example 1

Example 2

Example 3

Example 4

Example 5

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?

Summary

    Understanding Mutations Identifying Resistant Mutations Subtype Determination Phylogenetic Analysis

HIV Drug Resistance Training Module 6: Sequencing Interpretation and Analysis Topics     Understanding Mutations Identifying Resistant Mutations Phylogenetic Analysis Subtype Determination.

Transcript HIV Drug Resistance Training Module 6: Sequencing Interpretation and Analysis Topics     Understanding Mutations Identifying Resistant Mutations Phylogenetic Analysis Subtype Determination.