1. 2003Cumulative Number of AIDS deaths: North America 20,000 500,000 World3,000,00022,00,000 Total number of infected people: North America 940,000 1,200,000 World 40,000,00062,000,000 Number of new infections: North America 45,000 World5,000,000 Number of people cured of HIV<1 Number of infections prevented by vaccination<1 2003Cumulative Number of AIDS deaths: North America 20,000 500,000 World3,000,00022,00,000 Total number of infected people: North America 940,000 1,200,000 World 40,000,00062,000,000 Number of new infections: North America 45,000 World5,000,000 Number of people cured of HIV<1 Number of infections prevented by vaccination<1 HIV/AIDS in 2004* *UNAIDS, December, 2003

2. The HIV Replication Cycle Adsorption to CD4 receptor Entry via fusion Following coreceptor binding Reverse transcription Transcription Integration Tat Rev splicing Nef Gag-Pro-Pol Maturation Vpu Vif Assembly Budding

3. Important Properties of HIV 1.Infection requires CD4 protein on the surface of the cell as receptor. 1.Therefore can only infect CD4+ (“helper”) T cells and a few others. 2.Almost all infected cells die within a day or two after infection. 3.Infected CD4 cells make enough virus particles to infect about the same number of new cells (10-100 million). 4.Therefore, the infection in an individual persists by constant, repeated cycles of infection and cell death (about 1 a day). 5.These properties are also found in the benign SIV-monkey infections, but in humans there is a slow loss of total CD4 cells, leading eventually to failure of the immune system. 1.Infection requires CD4 protein on the surface of the cell as receptor. 1.Therefore can only infect CD4+ (“helper”) T cells and a few others. 2.Almost all infected cells die within a day or two after infection. 3.Infected CD4 cells make enough virus particles to infect about the same number of new cells (10-100 million). 4.Therefore, the infection in an individual persists by constant, repeated cycles of infection and cell death (about 1 a day). 5.These properties are also found in the benign SIV-monkey infections, but in humans there is a slow loss of total CD4 cells, leading eventually to failure of the immune system.

4. HIV RNA (copies/ml plasma) CD4 + T cells (cells/  l)

5. RNA Copies/ml Appearance of 3TC-Resistant Mutations in Treated Patients

7. Approximate num ber of infected cells Virus load (copies/ml) 8 7 6 5 4 3 2 1 0 Years

8. Zhang et al NEJM 340:1605-1613 Persistence of Cells latently Infected with HIV after Suppression of Viremia to “Undetectable” Levels

9. 0 200 400 600 800 Time (days) 10 7 10 7 10 6 10 6 10 5 10 5 10 4 10 4 10 3 10 3 10 2 10 2 10 1 10 1 10 0 10 0 10 -1 10 -1 0 200 400 600 800 Time (days) 10 7 10 7 10 6 10 6 10 5 10 5 10 4 10 4 10 3 10 3 10 2 10 2 10 1 10 1 10 0 10 0 10 -1 10 -1 Plasma HIV-1 RNA (copies/ml) 22 ± 6 c/ml 4 ± 2 c/ml Viremia Persists after Suppression by Antiretroviral Therapy Start Therapy (D4T/3TC/efavirenz) bDNA bDNA <75 copies/ml Single Copy Assay Single Copy Assay < 1 copy/ml

10. 1. After early primary infection, HIV gives lifelong persistent infection leading to AIDS after about 10 years (on average). 2. Persistence is due to constant replication of the virus and killing of 10 7 -10 9 infected CD4+ T cells at about 1 cycle/day. 3. Smaller fractions of “latently infected” cells that live much longer after infection are probably unimportant for the natural history of the infection, but very important for foiling treatment. 4. Constant replication day after day, year after year, leads to extensive genetic variation. Antigenic escape. Drug resistance. Variation in coreceptor usage. 5. The system remains in an extraordinarily robust quasi steady state for thousands of replication cycles before progressing to disease. 6. We still don’t know how HIV causes AIDS. 1. After early primary infection, HIV gives lifelong persistent infection leading to AIDS after about 10 years (on average). 2. Persistence is due to constant replication of the virus and killing of 10 7 -10 9 infected CD4+ T cells at about 1 cycle/day. 3. Smaller fractions of “latently infected” cells that live much longer after infection are probably unimportant for the natural history of the infection, but very important for foiling treatment. 4. Constant replication day after day, year after year, leads to extensive genetic variation. Antigenic escape. Drug resistance. Variation in coreceptor usage. 5. The system remains in an extraordinarily robust quasi steady state for thousands of replication cycles before progressing to disease. 6. We still don’t know how HIV causes AIDS. HIV-Host Interaction

11. HIV Drug Resistance 1.Introduction. 2.Mechanism of resistance. 3.Evolution of resistance. 1.Introduction. 2.Mechanism of resistance. 3.Evolution of resistance.

12. The HIV Replication Cycle Adsorption to CD4 receptor Entry via fusion Following coreceptor binding Entry via fusion Following coreceptor binding Reverse transcription Transcription Integration Tat Rev splicing Nef Gag-Pro-Pol Maturation Vpu Vif Assembly Budding

13. Unsuccessful (so far): Immunotherapy. Gene therapy. Recombinant antiviral proteins. Herbal extracts. Faith healing. Successful: Nucleoside RT inhibitors. (AZT, 3TC, ddI, ddC, d4T, etc.) Non nucleoside RT inhibitors. (Nevirapine, Efavirenz, Delavirdine,etc.) Protease inhibitors. (Indinavir, Saquinavir, Nelfinavir, Ritonavir, etc.) Fusion inhibitors (Enfuvirtide). Promising: Integrase inhibitors. Coreceptor inhibitors. Unsuccessful (so far): Immunotherapy. Gene therapy. Recombinant antiviral proteins. Herbal extracts. Faith healing. Successful: Nucleoside RT inhibitors. (AZT, 3TC, ddI, ddC, d4T, etc.) Non nucleoside RT inhibitors. (Nevirapine, Efavirenz, Delavirdine,etc.) Protease inhibitors. (Indinavir, Saquinavir, Nelfinavir, Ritonavir, etc.) Fusion inhibitors (Enfuvirtide). Promising: Integrase inhibitors. Coreceptor inhibitors. Anti-HIV Therapies

14. Protease Inhibitors: Nonnucleoside RT Inhibitors: Nucleoside RT Inhibitors: Nucleoside RT Inhibitors: Zidovudine (AZT) Zalcitabine (ddC) Nelfinavir Ritonavir Saquinavir Nelfinavir Ritonavir Saquinavir Stavudine (d4T) Tenofovir Stavudine (d4T) Tenofovir Lopinavir Nevirapine Lamivudine (3TC) Indinavir Efavirenz Didanosine (ddI) Amprenavir Delavirdine Abacavir Approved anti-HIV Drugs 2003 Fusion Inhibitors: Enfuvirtide (T20) Enfuvirtide (T20)

15. Deaths per 100 person-years Therapy with a protease inhibitor % of pa tient-days

16. 1. Occurs with all (effective) antivirals tested to date, in vivo and in vitro. (“If you don’t get resistance, the drug’s no good.”) 2. Is the most important factor preventing successful long term treatment. (If resistance did not arise, we would probably not be here today.) 3. Monotherapy almost always rapidly fails, most likely due to selection of mutants already present in the virus population. 4. Our only way to deal with this problem at present is to throw enough drugs at it so that no preexisting variant is resistant to all of them, and that replication is sufficiently suppressed to prevent further evolution. 5. A patient for whom this therapy has failed has very few treatment options left. 1. Occurs with all (effective) antivirals tested to date, in vivo and in vitro. (“If you don’t get resistance, the drug’s no good.”) 2. Is the most important factor preventing successful long term treatment. (If resistance did not arise, we would probably not be here today.) 3. Monotherapy almost always rapidly fails, most likely due to selection of mutants already present in the virus population. 4. Our only way to deal with this problem at present is to throw enough drugs at it so that no preexisting variant is resistant to all of them, and that replication is sufficiently suppressed to prevent further evolution. 5. A patient for whom this therapy has failed has very few treatment options left. HIV Drug Resistance

17. Location of Drug Resistant Mutations in HIV Proteins 0100200300400500 Nucleoside RT inhibitors Nonnucleoside RT inhibitors Pyrophosphate RT inhibitors Protease inhibitors Binding/fusion inhibitors A. Reverse transcriptase B. Protease D. Env FingersPalm Fingers PalmThumbConnectionRNase H Integrase inhibitors C. Integrase gp120SUgp41TM 600700800900 V1V2V3V4V5 C1 C2C3C4EctodomainCytoplasmic domain Amino Acid Position

18. Drug-Resistant Mutations in HIV 1.NNRTI’s 2.3TC 3.AZT 4.Protease inhibitors 1.NNRTI’s 2.3TC 3.AZT 4.Protease inhibitors

19. thumb fingers palm RNase H p66 p51 pol active site RNase H active site Courtesy of E. Arnold template primer

20. Locations of Drug Resistance Mutation Sites in HIV-1 RT/DNA Structure Nucleoside drug resistance mutation sites Non-nucleoside drug resistance mutation sites

21. NNRTI Resistance 1.Occurs rapidly in patients and cell culture. 2.Virtually complete cross-resistance to chemically very different compounds. 3.Mutations in binding “pocket” at base of thumb domain. 4.Pocket does not exist in the native structure. 1.Occurs rapidly in patients and cell culture. 2.Virtually complete cross-resistance to chemically very different compounds. 3.Mutations in binding “pocket” at base of thumb domain. 4.Pocket does not exist in the native structure.

22. Binding of NNRTIs to HIV-1 RT Courtesy of E. Arnold

23. Leu 100 Lys 101 Lys 102 Tyr 188 Tyr 181 Lys 103 Glu 138 (B) OW NNRTI binding pocket region in wild-type HIV-1 RT structure    p51 Courtesy of E. Arnold

24. Tyr 181 Tyr 188 Gly 190 Lys 103 Pro 95 Trp 229 Leu 100 Leu 234 Val 106 Pro 236 Phe 227 Tyr 318 Interactions are predominantly hydrophobic (with side- chains of L100, Y181, Y188, F227, W229, L234, and Y318). Inhibitor-protein Interactions Courtesy of E. Arnold

25. Cys 181 Tyr 188 Glu 138 (B) Asn 136 (B) Asn 103 Lys 102 Lys 101 Leu 100 OW NNRTI binding pocket region in K103N/Y181C mutant structure    2.8 Å 3.1 Å 3.3 Å Courtesy of E. Arnold

26. 3TC Resistance 1.Occurs rapidly in patients and cell culture. 2.Mutations nearly always at M184 in active site. 3.RT from resistant virus is resistant to 3TCTTP incorporation. 4.Resistance due to steric hindrance. 1.Occurs rapidly in patients and cell culture. 2.Mutations nearly always at M184 in active site. 3.RT from resistant virus is resistant to 3TCTTP incorporation. 4.Resistance due to steric hindrance.

27. Structure of 3TC Deoxycytidine3TC (-)-2´, 3´-dideoxy-3´-thiacytidine (3TC)

28. dTTP 3 TC Sarafianos, et al.,1999. PNAS USA 96: 10027-10032. Steric Hindrance in 3TC Resistance

29. AZT Resistance 1.Occurs rapidly in patients and cell culture. 2.Mutations not at active site or dNTP binding site. 3.RT from resistant virus not resistant to AZTTP incorporation. 4.Resistance due to excision (pyrophosphorolysis). 1.Occurs rapidly in patients and cell culture. 2.Mutations not at active site or dNTP binding site. 3.RT from resistant virus not resistant to AZTTP incorporation. 4.Resistance due to excision (pyrophosphorolysis).

30. Courtesy of S. Hughes

32. Clash Between Incorporated AZT and Active site Aspartic Acid Prevents Translocation Courtesy of S. Hughes

33. Other N RTI AZT Courtesy of S. Hughes

34. Protease Inhibitor Resistance 1.Occurs rapidly in patients and cell culture. 2.Usually associated with multiple mutations that arise sequentially. 3.Initial (primary) mutations at or near active site, and often greatly reduce fitness. 4.Subsequent mutations often far away from active site (or even in gag), and, in many cases, act to improve fitness. 1.Occurs rapidly in patients and cell culture. 2.Usually associated with multiple mutations that arise sequentially. 3.Initial (primary) mutations at or near active site, and often greatly reduce fitness. 4.Subsequent mutations often far away from active site (or even in gag), and, in many cases, act to improve fitness.

35. Courtesy of A. Wlodawer

36. Time (weeks) Relative virus load Primary mutation Secondary mutations Evolution of Protease Inhibitor Resistance in Vivo

37. Modeling HIV Variation How do drug resistance mutations arise?

38. Time, Weeks Virus Load Start 3TC Evolution of 3TC Resistance

39. M184V (AUG-GUG) Start 3TC M184 (AUG) M184I (AUG-AUA) Time, Weeks Virus Load Evolution of 3TC Resistance

40. Start 3TC M184 (AUG) M184I (AUG-AUA) M184V (AUG-GUG) Time, Weeks Virus Load Evolution of 3TC Resistance

41. Evolution of HIV in Infected People Thesis: 1. Drug-resistant mutations are present in the virus population prior to therapy. 2. In the absence of drug, these mutations are slightly deleterious to the virus. Thesis: 1. Drug-resistant mutations are present in the virus population prior to therapy. 2. In the absence of drug, these mutations are slightly deleterious to the virus.

42. Factors of HIV Evolution 1. Mutation

43. Factors of HIV Evolution More fit Less fit 2. Selection

44. Factors of HIV Evolution More important in small populations of cells Mutant proportion changes due to random sampling 3. Drift

45. Factors of HIV Evolution Etc. Selection at linked sites is not independent, even in large populations. Deleterious mutations accumulate. Can be reversed by recombination or compensating mutations. 4. Linkage

46. Mutant frequency.03.02.01 1 /  Ns 1/s 0 Large Medium Accumulation of Drug Resistant Mutations Before Start of Therapy 0 0.01 0.02 0.03 Time, generations (years after infection) 0100020003000 Small (0)(5)(10)(15) Large, Linkage Population size:

47. Large, Linkage Large, linkage Plus recombination or compensating mutations Mutant frequency.03.02.01 1 /  Ns 1/s 0 Accumulation of Drug Resistant Mutations Before Start of Therapy 0 0.01 0.02 0.03 Time, generations (years after infection) 0100020003000 (0)(5)(10)(15) Population size:

48. Genetics of HIV-1 Populations in Infected Individuals National Cancer Institute at Frederick HIV Drug Resistance Program

49. To obtain a detailed analysis of HIV-1 genetic diversity in infected patients to understand: Roles of mutation and selection. Roles of mutation and selection. Replicating population size. Replicating population size. Extent of recombination. Extent of recombination. Presence of compensatory mutations. Presence of compensatory mutations. Objectives

50. Patients in Study 1. Recent HIV infection (n=3)1. Recent HIV infection (n=3) –10-100 days post-infection –VL=12,000->500,000 copies/ml 2. Chronic untreated HIV infection (n=3)2. Chronic untreated HIV infection (n=3) –>4 mos-10 years –VL=7,300-30,000 copies/ml 3. Chronic HIV infection initiating ART (n=3)3. Chronic HIV infection initiating ART (n=3) –>6 mos-15 years –VL=86,000-1,500,000 copies/ml

51. Limiting Dilution PCR Sequencing AKA: single genome sequencing (SGS) p6 PRRTAmplicon gag pro pol Sequence Analysis PCR Dilute to 30% positive To cDNA Plasmavirus

52. Assay Characteristics Amplicon includes nearly all major sites for resistance mutations Amplicon includes nearly all major sites for resistance mutations Variation less than env, but gives adequate phylogenetic signal Variation less than env, but gives adequate phylogenetic signal Does not give alignment problems Does not give alignment problems Background: Background: error rate = 0.012% recombination rate < 1 per 60,000 nucleotides

53. Nucleotide Substitutions, % 0 5.5 24 HXB2 clippedp6prrt 0706-14 0706-15 0706-16 0706-17 0706-2 0706-10 NG-5 NG-7 NG-6 NG-10 NG-2 NG-11 SR-4 SR-5 SR-3 SR-2 SR-6 SR-11 BP-16 BP-3 BP-4 BP-7 HXB2 Recent HIV Infection Patient 3 Typical Chronic Patient Patient 2 Patient 1

54. Patients in Study 1. Recent HIV infection (n=3)1. Recent HIV infection (n=3) –10-100 days post-infection –VL=12,000->500,000 copies/ml 2. Chronic untreated HIV infection (n=3) –>4 mos-10 years –VL=7,300-30,000 copies/ml 3. Chronic HIV infection initiating ART (n=3) –>6 mos-15 years –VL=86,000-1,500,000 copies/ml

55. s=0.05 s=0.1 s=0.15 s=0.2 Purifying selection: 0.025.050.075.100.125.150.175 Days after infection Mutation frequency (%) Accumulation of Mutations Early after Infection.200 0102030405060 s=0 (Neutral accumulation)  =3x10 -5 substitutions/cycle 1.14 cycles/day (measured for patient 1) 70 Assay background=0.012%

56. Conclusions: Recently Infected Patients  Virus populations were nearly homogenous  Mutation frequencies for most patients were not distinguishable from the error rate of the limiting dilution assay and well below those expected for neutral accumulation.  Strong purifying selection on HIV-1 populations early during HIV infection?

57. Patients in Study 1. Recent HIV infection (n=3) –10-100 days post-infection –VL=12,000->500,000 copies/ml 2. Chronic untreated HIV infection (n=3)2. Chronic untreated HIV infection (n=3) –>4 mos-10 years –VL=7,300-30,000 copies/ml 3. Chronic HIV infection initiating ART (n=3) –>6 mos-15 years –VL=86,000-1,500,000 copies/ml

58. HXB2 Months Viral Load Patient 1 42 y.o. male HIV infection>10 y CD4=348 cells/µl HIV Diversity over Time in Chronic Infection 0 4 8 12 16 20 10 6 10 4 10 2 10 0 No discernable change in tree over nearly 2 years.

59. 3.1 0 HXB2 Start Therapy Antiretroviral Therapy Initiated Patient 3 45 yo male HIV infection >15 y CD4=111 cells/  l Similar HIV variants present after >2 log decrease in viral load 0 2 4 6 8 10 12 Weeks 10 10 3 10 5 10 7 Virus load

60. Conclusions: Chronic Untreated Infection Diverse population: no two sequences are the same with the average intrapatient diversity of nearly 2%. No evident shift in population structure over 18 months. No evident shift in population structure over 18 months. Extensive recombination. Extensive recombination. Evidence for compensatory mutations. Evidence for compensatory mutations. Implies a large, genetically stable population. Implies a large, genetically stable population.

61. 1. The genetic structure of HIV populations, as measured by diversity in pro and pol, is constant over both long periods of time and large decreases in viral load. 3. These results are consistent with a very large population, replicating at a genetic balance, and subject to strong purifying selection, implying that resistance may be predictable. Overall Conclusions: