2003Cumulative Number of AIDS deaths: North America 20,000 500,000

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.



Adsorption to CD4 receptor Adsorption to CD4 receptor



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.


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


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


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


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


3TC Resistance3TC Resistance


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.


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


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).


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


Clash Between Incorporated AZT and Active site Aspartic Acid Prevents

Translocation


Other

NRTI

AZT


Protease Inhibitor ResistanceProtease Inhibitor Resistance


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.


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


More important in small populations of cells

Mutant proportion changes due to random sampling

3. Drift


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


• 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?


• 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:

Documents

2003Cumulative Number of AIDS deaths: North America 20,000 500,000