K103 what ? Making Sense of Antiretroviral Drug Resistance Testing

1. K103 what?Making Sense of AntiretroviralDrug Resistance Testing DPET 810 Christopher Hurt, MD February 5, 2008

2. Outline • A little HIV history • HIV replication cycle • Why HIV gets resistant • Testing: recommendations and methodologies • Phenotyping, genotyping, and “virtual” phenotypes • The importance of being conservative… if you’re an enzyme • Real-life examples (time permitting)

3. HIV • Two distinct retroviruses, HIV-1 and HIV-2 • HIV-1 predominates • HIV-2 seen mainly in Africa • First (retrospectively) confirmed case from Congo in 1959(1) • First 5 reported cases in June 1981(2) • MSM with PCP in LA • Association of AIDS cases with virus confirmed in 1984(3) 1Nature 1998:391:594-7 2MMWR 1981:30:250-2 3Science 1984:224:500-3

4. HIV replication and drug targets

5. HIV can’t read what it writes • Cellular DNA polymerases can “proofread” • 3’ exonuclease function • High-fidelity replication • RT lacks exonuclease activity • Sloppy proofreading = lots of mutations (µ) • Infected cell lives ~1-2d; HIV generation time 2.6d(4) • 109 virions must be released daily to maintain viremia(4) • 106 to 107 proviruses, but aren’t actively replicating • Each and every possible point µ occurs 104 to 105 times each day in an untreated HIV+ person(5) 4Science 1996:271:1582-6 5Science 1995;267:483-9

6. Only the fittest survive • Darwinian evolution at work • ARVs apply selection pressure to viral population • Bottleneck effect • Remember all mutants are archived!

7. Transmitted drug resistance (TDR) • First described in 1993 • Sexual transmission of ZDV resistant virus(6) • Overall TDR prevalence approx 10-15%(7) • Large European, US national samples Graph from (8) 6NEJM 1993:328:1163-5 7Antivir Ther 2004;9:695-702 8JAMA 2002;288:181-8

8. TDR in North Carolina • Overall prevalence ~17% • NRTIs, 4-5% • NNRTIs, 9-10% • PIs, 3-4% • NNRTI resistance increasing over time(9) 9Antivir Ther 2007;12:S55

9. Who gets tested? • DHHS Guidelines, 2007 • All HIV+ patients entering care, regardless of whether therapy initiated immediately(A-III) • Genotypic assays preferred for ARV naïves(A-III) • At virologic failure(A-II) • Suboptimal viral load reduction(A-II)

10. Just one catch… • Assays rely on population sequencing • Lower LOD for any quasispecies is ~20% of total population • If you stop the meds,the proportion of resistantvirus in the population drops • For test to be useful, must beon meds (or very recently off them)

11. RT and PR genes from pt’s HIV RNA amplified by RT-PCR (gag-pol) Amplicon + luciferase reporter gene = vector Cells co-transfected with vector + env gene Virions harvested Target cells infected Luciferase activity measured (less is better!) Phenotypic testing RTIs added here PIs added here

12. Phenotypic testing • Susceptibility measured by comparing reporter expression ± ARVs • Percent of inhibition of activity plotted against log10 of [ARV] • Curve used to calculate IC50 • Conc’n needed for 50% reduction in replication • Pt’s IC50 compared with IC50 of ARV-sensitive control strain, to determine fold-change • Also yields a replicative capacity (RC) measurment

13. ZDV EFV RC TPV

14. Advantages Detailed in vitro information May show subtleties of your pt’s virus RC measured Combinations of drugs can be assessed for synergy Disadvantages Turnaround = 2-4 wks Lab and labor-intensive and  very expensive Unclear what IC50 = clinical failure vs in vitro failure Most (but not all) of gag-pol is amplified Theoretical skewing of PI results Phenotypic testing

15. Genotypic testing • Pt’s gag-pol amplified from serum by RT-PCR • Almost all of PR, at least positions 41-236 of RT • Dideoxynucleotidesequencing • dNTP + ddNTPmixture • Chain termination • Electrophoresisseparates outdifferent lengths • Automated readingof sequence

16. Genotypic testing • DNA sequence product aligned based on conserved areas, then read out as codons • Amino acid sequence interpreted from codons • Comparison made to published lists of mutations from Int’l AIDS Society-USA (IAS)

17. Advantages Exact information about the mutations present Specific µ elicited by particular ARVs are well-described, predictable Relatively fast (turnaround = 7-10 d) Disadvantages Result is an ordered string of letters (e.g., NRKLPDS) or A#Bs (e.g., M41L, K103N) Interpretation requires knowledge of µ and their clinical relevance Also cross-resistance among different drugs Genotypic testing

18. Caveats • Both tests start off with a PCR-based amplification • Pretty good, but not perfect… • Minority quasispecies are missed • May be clinically significant • If patient is off meds, utility of results are anybody’s guess • Except in the case of TDR…

19. “Virtual phenotyping” • Genotyping yields DNA, amino acid sequences • Repositories of paired genotypes and phenotypes administered by companies (e.g., Virco) • Correlation between pt’s sequence and database info yields a predicted response to particular ARVs • Can’t describe synergy between ARVs… yet

20. VircoTYPE • Assay used at UNC • Curves constructed drug-by-drug • “Clinical cutoffs” (CCOs) set between 20% and 80% loss of wild-type susceptibility • “Biological cutoffs” (BCOs) set for drugs without well-defined ranges

25. Why these positions?

26. Protein structure • Proteins fold after translation from RNA • Primary structure • Amino acid sequence • Secondary structure • Alpha helices and beta sheets • Tertiary structure • Domains, etc.

27. Reverse transcriptase • “Right hand” shape • Fingers, thumb wrap around RNA/DNA • Palm contains active site • D110, D185, D186

28. Finger and palm domains Active siteresidues

30. M184V K65R D67N K70R M41L K103N K219Q T215YF L210W

31. M184V K65R D67N K70R M41L K103N K219Q T215YF L210W

32. M184V K65R D67N K70R M41L K103N K219Q T215YF L210W

33. M184V K65R D67N K70R M41L K103N K219Q T215YF L210W

34. M184V K65R D67N K70R M41L K103N K219Q T215YF L210W

35. Protease • Two “flaps” of homodimer allow HIV polypeptide to access active site at center:D25, T26, G27 • Pocket regions act like recesses of a key, recognizing portions of polypeptide D67N V82 I84V Pocket V32I L33F M46L I47V I50V Flap domain I54V

36. So why those positions? • These are spots where variability of the enzyme’s amino acid sequence won’t affect its general functionality or tertiary structure • Protein sequences are highly conserved where they need to be, and variable where they can be • With key exceptions (e.g., K103N, TAMs), you need to accumulate multiple mutations before significant resistance develops

37. Take-home points • All patients coming into care for the first time need an assessment of their ARV resistance • About 1:10 to 1:20 newly infected patients will be resistant to some drug at baseline • ARVs are a selection pressure, and the composition of the viral swarm determines how useful resistance test results are • TEST THEM WHILE STILL ON THEIR MEDS!!! • Phenotyping gives you in vitro data and an IC50, but won’t tell you what mutations made it resistant

38. Take-home points • Genotyping gives you a mutation list which you have to interpret and apply clinically • Virtual phenotyping correlates a patient’s sequence with a known phenotype, and is usually very concordant • Position numbers for AAs correspond with a three-dimensional enzyme structure • Some individual µ are advantageous (M184V) and others are potentially disastrous (K103N, TAMs)