2003cumulative number of aids deaths: north america 20,000 500,000
DESCRIPTION
HIV/AIDS in 2004*. 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: - PowerPoint PPT PresentationTRANSCRIPT
2003 CumulativeNumber of AIDS deaths:
North America 20,000 500,000World 3,000,000 22,00,000
Total number of infected people:North America 940,000 1,200,000World 40,000,000 62,000,000
Number of new infections:North America 45,000World 5,000,000
Number of people cured of HIV <1
Number of infections prevented by vaccination <1
2003 CumulativeNumber of AIDS deaths:
North America 20,000 500,000World 3,000,000 22,00,000
Total number of infected people:North America 940,000 1,200,000World 40,000,000 62,000,000
Number of new infections:North America 45,000World 5,000,000
Number of people cured of HIV <1
Number of infections prevented by vaccination <1
HIV/AIDS in 2004*HIV/AIDS in 2004*
*UNAIDS, December, 2003*UNAIDS, December, 2003
The HIV Replication Cycle
Adsorption to CD4 receptor
Entry via fusionFollowing coreceptor binding
Reverse transcription
Transcription
Integration
TatRev
splicing
Nef
Gag-Pro-Pol
Maturation
Vpu
Vif
AssemblyBudding
Important Properties of HIVImportant 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.
"Typical" Course of HIV Infection
HIV
RN
A (
cop
ies/
ml
pla
sma)
1200
1000
800
600
400
200
0
107
106
105
104
103
1020 3 6 9 12 2 4 6 8 10 12
Weeks YearsTime after infection
Primaryinfection
Acute HIV infectionWide distribution of virusSeeding of lymphoid organs
Clinical latency
Onset ofsymptoms
Opportunisticinfections
Death
Appearance ofanti-HIV CTL's
CD
4+ T
cel
ls (
cell
s/l
)
100,000
75,000
50,000
25,000
00 1 2 3 4 5 6 7 8 9 10 11 12
Weeks after start of 3TC
Wild type at codon 184
M184V
M184I
RN
A C
op
ies/
ml
Appearance of 3TC-Resistant Mutations in Treated Patients
4 Populations of HIV Infected Cells
0
-1
-2
-3
Virus Load
T1/2 ~ 2 Days
T1/2 ~ 20 Days
T1/2 ~ 100 Days
Initiate suppressive antiviral therapy
0 2 4 6 8
Weeks of Treatment
LogRelativeValues
Viral DNA Positive Cells
T1/2 ~ 100(?) Days
Quiescent infected CD4 cells
10
Ap
pro
xim
ate
nu
mb
er o
f in
fect
ed c
ells
Vir
us
load
(co
pie
s/m
l)
8
7
6
5
4
3
2
1
0
Years
105
104
103
102
101
100
10-1
10-1
10-3
10
10
10
10
10
10
10
10
10
0 1 2 3
"Undetectable"
"Eradication"
Is Eradication Possible?
Zhang et al NEJM 340:1605-1613
Persistence of Cells latently Infected with HIV after Suppression of Viremia to “Undetectable” Levels
0 200 400 600 0 200 400 600 800800 Time (days)Time (days)
101077
101066
101055
101044
101033
101022
101011
101000
1010-1-1
0 200 400 600 0 200 400 600 800800 Time (days)Time (days)
101077
101066
101055
101044
101033
101022
101011
101000
1010-1-1
Pla
sma
HIV
-1 R
NA
(co
pie
s/m
l)P
lasm
a H
IV-1
RN
A (
cop
ies/
ml)
22 ± 6 c/ml 22 ± 6 c/ml 4 ± 2 c/ml4 ± 2 c/ml
Viremia Persists after Suppression by Viremia Persists after Suppression by Antiretroviral TherapyAntiretroviral Therapy
Viremia Persists after Suppression by Viremia Persists after Suppression by Antiretroviral TherapyAntiretroviral Therapy
Start Therapy (D4T/3TC/efavirenz)Start Therapy (D4T/3TC/efavirenz) Start Therapy (D4T/3TC/efavirenz)Start Therapy (D4T/3TC/efavirenz)
bDNAbDNA
bDNA <75 copies/mlbDNA <75 copies/ml
Single Copy AssaySingle Copy Assay
Single Copy Assay < 1 copy/ml Single Copy Assay < 1 copy/ml
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 107-109 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 107-109 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 InteractionHIV-Host Interaction
HIV Drug ResistanceHIV Drug Resistance
1. Introduction.
2. Mechanism of resistance.
3. Evolution of resistance.
1. Introduction.
2. Mechanism of resistance.
3. Evolution of resistance.
The HIV Replication Cycle
The HIV Replication Cycle
Adsorption to CD4 receptor Adsorption to CD4 receptor
Entry via fusionFollowing coreceptor binding
Entry via fusionFollowing coreceptor binding
Reverse transcription
Transcription
Integration
TatRev
splicing
Nef
Gag-Pro-Pol
MaturationMaturation
Vpu
Vif
AssemblyBudding
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 TherapiesAnti-HIV Therapies
Protease Inhibitors:Protease Inhibitors:
Nonnucleoside RT Inhibitors:Nonnucleoside RT Inhibitors:
Nucleoside RTInhibitors: Nucleoside RTInhibitors:
Zidovudine (AZT)Zidovudine (AZT)
Zalcitabine (ddC)Zalcitabine (ddC)
Nelfinavir
Ritonavir
Saquinavir
Nelfinavir
Ritonavir
Saquinavir
Stavudine (d4T)
Tenofovir
Stavudine (d4T)
Tenofovir
LopinavirLopinavirNevirapineNevirapineLamivudine (3TC)Lamivudine (3TC)
IndinavirIndinavirEfavirenzEfavirenzDidanosine (ddI)Didanosine (ddI)
AmprenavirAmprenavirDelavirdineDelavirdineAbacavirAbacavir
Approved anti-HIV Drugs2003
Approved anti-HIV Drugs2003
Fusion Inhibitors:Fusion Inhibitors:Enfuvirtide
(T20)
Enfuvirtide
(T20)
Dea
ths
per
100
per
son
-yea
rs
Th
erap
y w
ith
a p
rote
ase
inhi
bit
or
% o
f pat
ien
t-d
ays
1994 1995 1996 1997
40
30
20
10
0
100
80
60
40
20
0
Year
Mortality and Protease Inhibitor Use in HIV-Infected Patients
Pallella, et alNew Engl. J. Med.338:853. 1998
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 ResistanceHIV Drug Resistance
Location of Drug Resistant Mutations in HIV Proteins
0 100 200 300 400 500
Nucleoside RT inhibitors
Nonnucleoside RT inhibitorsPyrophosphate RT inhibitorsProtease inhibitors
Binding/fusion inhibitors
A. Reverse transcriptase
B. Protease
D. Env
Fingers PalmFingers
Palm Thumb Connection RNase H
Integrase inhibitors
C. Integrase
gp120SU gp41TM
600 700 800 900
V1 V2 V3 V4 V5C1 C2 C3 C4 Ectodomain Cytoplasmic domain
Amino Acid Position
Drug-Resistant Mutations in HIVDrug-Resistant Mutations in HIV
1. NNRTI’s
2. 3TC
3. AZT
4. Protease inhibitors
1. NNRTI’s
2. 3TC
3. AZT
4. Protease inhibitors
thumb
fingers
palm
RNase H
p66
p51
pol active site
RNase H
active site
Courtesy of E. Arnold
template
primer
Locations of Drug Resistance MutationSites in HIV-1 RT/DNA Structure
Nucleoside drug resistance mutation sitesNon-nucleoside drug resistance mutation sites
NNRTI ResistanceNNRTI 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.
Binding of NNRTIs to HIV-1 RT
Courtesy of E. Arnold
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
Tyr 181 Tyr 188
Gly 190
Lys 103
Pro 95 Trp 229Leu 100
Leu 234
Val 106
Pro 236Phe 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
Cys 181 Tyr 188
Glu 138 (B)
Asn 136 (B)
Asn 103
Lys 102
Lys 101Leu 100
OW
NNRTI binding pocket region in K103N/Y181C mutant structure
2.8 Å
3.1 Å 3.3 Å
Courtesy of E. Arnold
3TC Resistance3TC 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.
N
N
O
NH2
S
OOH
O
N
N
O
NH2
OH
OH
Structure of 3TC
Deoxycytidine 3TC
(-)-2´, 3´-dideoxy-3´-thiacytidine (3TC)
dTTP 3TC
Sarafianos, et al.,1999. PNAS USA 96: 10027-10032.
Steric Hindrance in 3TC Resistance
AZT ResistanceAZT 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).
Courtesy of S. Hughes
Courtesy of S. Hughes
Clash Between Incorporated AZT and Active site Aspartic Acid Prevents
Translocation
Courtesy of S. Hughes
Other
NRTI
AZT
Courtesy of S. Hughes
Protease Inhibitor ResistanceProtease 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.
Courtesy of A. WlodawerCourtesy of A. Wlodawer
Time (weeks)
Rel
ativ
e vi
rus
load
Primarymutation
Secondarymutations
Evolution of Protease Inhibitor Resistance in Vivo
Modeling HIV VariationModeling HIV Variation
How do drug resistance mutations arise?How do drug resistance mutations arise?
Time, Weeks
Vir
us
Lo
ad
Start 3TC
Evolution of 3TC Resistance
M184V (AUG-GUG)
Start 3TC
M184 (AUG)
M184I (AUG-AUA)
Time, Weeks
Vir
us
Lo
adEvolution of 3TC Resistance
Start 3TC
M184 (AUG)
M184I (AUG-AUA)
M184V (AUG-GUG)
Time, Weeks
Vir
us
Lo
adEvolution of 3TC Resistance
Evolution of HIV in Infected PeopleEvolution 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.
Factors of HIV Evolution1. Mutation
Factors of HIV Evolution
More fit
Less fit
2. Selection
Factors of HIV Evolution
More important in small populations of cells
Mutant proportion changes due to random sampling
3. Drift
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
Mu
tan
t fr
equ
ency
.03
.02
.01
1/Ns1/s
0Large
Medium
Accumulation of Drug Resistant Mutations Before Start of Therapy
0
0.01
0.02
0.03
Time, generations(years after infection)
0 1000 2000 3000
Small
(0) (5) (10) (15)
Large,Linkage
Population size:
Large,Linkage
Large,linkage
Plus recombinationor compensating mutations
Mu
tan
t fr
equ
ency
.03
.02
.01
1/Ns1/s
0
Accumulation of Drug Resistant Mutations Before Start of Therapy
0
0.01
0.02
0.03
Time, generations(years after infection)
0 1000 2000 3000(0) (5) (10) (15)
Population size:
Genetics of HIV-1 Populations Genetics of HIV-1 Populations in Infected Individualsin Infected Individuals
National Cancer Institute at Frederick
HIV Drug Resistance ProgramHIV Drug Resistance Program
To obtain a detailed analysis of HIV-1 genetic To obtain a detailed analysis of HIV-1 genetic diversity in infected patients to understand: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.
ObjectivesObjectives
Patients in StudyPatients 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
Limiting Dilution PCR SequencingLimiting Dilution PCR SequencingAKA: single genome sequencing (SGS)AKA: single genome sequencing (SGS)
p6p6 PRPR RTRT
AmpliconAmplicon
gag pro polgag pro pol
Sequence Sequence
AnalysisAnalysis
PCRPCR
Dilute to 30% positiveDilute to 30% positiveTo To cDNAcDNA
PlasmaPlasmavirusvirus
Assay CharacteristicsAssay Characteristics
• • Amplicon includes nearly all major sites for Amplicon includes nearly all major sites for resistance mutations resistance mutations
• • Variation less than Variation less than envenv, but gives adequate , but gives adequate
phylogenetic signalphylogenetic signal
• • Does not give alignment problemsDoes not give alignment problems
• • Background:Background:error rate = 0.012% error rate = 0.012% recombination rate < 1 per 60,000 nucleotides recombination rate < 1 per 60,000 nucleotides
Nucleotide Substitutions, %
05.5
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
HXB2Recent Recent HIV InfectionHIV Infection Patient 3
TypicalChronicPatient
Patient 2
Patient 1
Patients in StudyPatients in Study
• 1. Recent HIV infection (n=3)1. Recent HIV infection (n=3)– 10-100 days post-infection10-100 days post-infection– VL=12,000->500,000 copies/mlVL=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
s=0.05
s=0.1s=0.15s=0.2
Purifying selection:
0
.025
.050
.075
.100
.125
.150
.175
Days after infection
Mu
tati
on
fre
qu
ency
(%
)Accumulation of Mutations Early
after Infection.200
0 10 20 30 40 50 60
s=0(Neutral accumulation)
=3x10-5 substitutions/cycle1.14 cycles/day (measured for patient 1)
70
Assay background=0.012%
Conclusions:Conclusions:Recently Infected PatientsRecently Infected Patients
Virus populations were nearly Virus populations were nearly homogenoushomogenous
Mutation frequencies for most patients Mutation frequencies for most patients were not distinguishable from the error were not distinguishable from the error rate of the limiting dilution assay and well rate of the limiting dilution assay and well below those expected for neutral below those expected for neutral accumulation.accumulation.
Strong purifying selection on HIV-1 Strong purifying selection on HIV-1 populations early during HIV infection?populations early during HIV infection?
Patients in StudyPatients 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>4 mos-10 years– VL=7,300-30,000 copies/mlVL=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
HXB2
Months
Vir
al L
oad
Patient 142 y.o. maleHIV infection>10 yCD4=348 cells/µl
HIV Diversity over Time in HIV Diversity over Time in Chronic InfectionChronic Infection
0 4 8 12 16 20
106106
104
102
100
No discernable change in tree over nearly 2 years.
3.1 0HXB2
Start Therapy
Antiretroviral Therapy InitiatedAntiretroviral Therapy Initiated
Patient 345 yo maleHIV infection >15 yCD4=111 cells/l
Similar HIV variants present Similar HIV variants present after >2 log decrease in viral loadafter >2 log decrease in viral load
0 2 4 6 8 10 12
Weeks
10
103
105
107
Vir
us lo
ad
Conclusions: Conclusions: Chronic Untreated Infection Chronic Untreated Infection
• Diverse population: no two sequences are the Diverse population: no two sequences are the same with the average intrapatient diversity of same with the average intrapatient diversity of nearly 2%.nearly 2%.
• • No evident shift in population structure over 18 No evident shift in population structure over 18 months.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.
1. The genetic structure of HIV populations, 1. The genetic structure of HIV populations, as measured by diversity in as measured by diversity in propro and and polpol, is , is constant over both long periods of time constant over both long periods of time and large decreases in viral load.and large decreases in viral load.
3. These results are consistent with a very 3. These results are consistent with a very
large population, replicating at a genetic large population, replicating at a genetic balance, and subject to strong purifying balance, and subject to strong purifying selection, implying that resistance may be selection, implying that resistance may be predictable. predictable.
Overall Conclusions: Overall Conclusions: