HIV Diagnosis and Pathogenesis

Scott M. Hammer, M.D.

HIV Diagnosis

• Consider in anyone presenting with symptoms and signs compatible with an HIV-related syndrome or in an asymptomatic person with a risk factor for acquisition

• Full sexual and behavioral history should be taken in all patients- Assumptions of risk (or lack thereof) by clinicians are

unreliable

Laboratory Diagnosis of Established HIV Infection: Antibody Detection

• Screening- Serum ELISA- Rapid blood or salivary Ab tests

• Confirmation- Western blot

• Written consent for HIV Ab testing must be obtained and be accompanied by pre- and post-test counselling

Laboratory Diagnosis of Acute HIV-1 Infection

• Patients with acute HIV infection may present to a health care facility before full antibody seroconversion- ELISA may be negative

- ELISA may be positive with negative or indeterminant Western blot

• Plasma HIV-1 RNA level should be done if acute HIV infection is suspected

• Follow-up antibody testing should be performed to document full seroconversion (positive ELISA and WB)

HIV-1 Virion

HIV LifeCycle

Tat = transcriptional activatorRev = regulator of mRNA nuclear export

HIV-1: Genetic Organization

Established HIV Infection:Pathogenesis

• Active viral replication present throughout course of disease• Major reservoirs of infection exist outside of blood

compartment- Lymphoreticular tissues- Central nervous system- Genital tract

• Virus exists as multiple quasispecies- Mixtures of viruses with differential phenotypic and genotypic

characteristics may coexist

• At least 10 X 109 virions produced and destroyed each day• T1/2 of HIV in plasma is <6 h and may be as short as 30

minutes• Immune response, chemokine receptor status and HLA type

are important codeterminants of outcome

Determinants of Outcome:Selected Viral Factors

• Escape from immune response- Under immune selective pressure (cellular and humoral),

mutations in gag, pol and env may arise

• Attenuation- nef deleted viruses associated with slow or long-term

nonprogression in case reports and small cohorts

• Tropism- R5 to X4 virus conversion associated with increased viral

pathogenicity and disease progression

• Subtypes- Potential for varied subtypes to exhibit differential

transmissibility and virulence» Potential for greater heterosexual spread of some subtypes

Host Factors in HIV Infection (I)

• Cell-mediated immunity- Cytotoxic T cells

» Eliminate virus infected cells

» Play prominent role in control of viremia, slowing of disease progression and perhaps prevention of infection

- T-helper response» Vital for preservation of CTL response

• Humoral immunity- Role in prevention of transmission and disease

progression unclear

Role of CTL’s in Control of Viremia

Letvin N & Walker B: Nature Med 2003;9:861-866

Host Factors in HIV Infection (II)

• Chemokine receptors- CCR5-Δ32 deletion

» Homozygosity associated with decreased susceptibility to R5 virus infection

» Heterozygosity associated with delayed disease progression

- CCR2-V64I mutation» Heterozygosity associated with delayed disease

progression

- CCR5 promoter polymorphisms» 59029-G homozygosity associated with slower disease

progression» 59356-T homozygosity associated with increased perinatal

transmission

Host Factors in HIV Infection (III)

• Other genetic factors- Class I alleles B35 and Cω4

» Associated with accelerated disease progression

- Heterozygosity at all HLA class I loci» Appear to be protective

- HLA-B57, HLA-B27, HLA-Bω4, HLA-B*5701 » Associated with long-term non-progression

- HLA-B14 and HLA-C8» ?Associated with long-term nonprogression

Mechanisms of CD4+ Cell Death in HIV Infection

• HIV-infected cells- Direct cytolytic effect of HIV- Lysis by CTL’s- Apoptosis

» Potentiated by viral gp120, Tat, Nef, Vpu

• HIV-uninfected cells- Apoptosis

» Release of gp120, Tat, Nef, Vpu by neighboring, infected cells

- Activation induced cell death

The Variable Course of HIV-1 InfectionTypical Progressor Rapid Progressor

Vir

al R

eplic

atio

n

CD

4 Level

months years

Primary HIVInfection Clinical Latency AIDS

A Vir

al R

eplic

atio

n

CD

4 Level

months years

Primary HIVInfection AIDS

BV

iral

Rep

licat

ion C

D4 L

evel

months years

Primary HIVInfection Clinical Latency

C

Nonprogressor

?

Reprinted with permission from Haynes. In: DeVita et al, eds. AIDS: Etiology, Treatment and Prevention.4th ed. Lippincott-Raven Publishers; 1997:89-99.

Phases of Decay Under the Influence of Potent Antiretroviral Therapy

2-4 16-24

Time (weeks)

Ch

an

ge

in H

IV R

NA

(lo

g1

0)

0

-1

-2

T1/2 = 1 d (productively infected CD4’s)

T1/2 = 2-4 wks (macrophages, latently infected CD4’s,

release of trapped virions) T1/2 = 6-44 mos (resting,memory CD4’s)

Therapeutic Implications of First and Second Phase HIV RNA Declines

• Antiviral potency can be assessed in first 7-14 days- Should see 1-2 log declines after initiation of therapy in

persons with drug susceptible virus who are adherent

• HIV RNA trajectory in first 1-8 weeks can be predictive of subsequent response- Durability of response translates into clinical benefit

Phases of Decay Under the Influence of Potent Antiretroviral Therapy

2-4 16-24

Time (weeks)

Ch

an

ge

in H

IV R

NA

(lo

g1

0)

0

-1

-2

T1/2 = 1 d (productively infected CD4’s)

T1/2 = 2-4 wks (macrophages, latently infected CD4’s,

release of trapped virions) T1/2 = 6-44 mos (resting,memory CD4’s)

+Ag

-Ag

Restingnaïve

CD4+ T cell

ActivatedCD4+ T

cell

-Ag

ActivatedCD4+ T

cell

+Ag

RestingmemoryCD4+ T

cell

+Ag +Ag

Postintegration Latency

Preintegration Latency

Model of Post-Integration Latency

Siliciano R et al

Therapeutic Implications of Third Phase of HIV RNA Decay: Latent Cell Reservoir

• Viral eradication not possible with current drugs• Archive of replication competent virus history is

established- Drug susceptible and resistant

• Despite the presence of reservoir(s), minimal degree of viral evolution observed in patients with plasma HIV RNA levels <50 c/ml suggests that current approach designed to achieve maximum virus suppression is appropriate

Initiation of Therapy in Established HIV Infection: Considerations

• Patient’s disease stage- Symptomatic status- CD4 cell count- Plasma HIV-1 RNA level

• Patient’s commitment to therapy

• Philosophy of treatment- Pros and cons of ‘early’ intervention

Initiation of Therapy in Asymptomatic Persons: Population Based Studies

• Clinical outcome compromised if Rx begun when CD4 <200- Miller et al (EuroSIDA), Ann Intern Med 1999;130:570-577- Hogg et al (British Columbia), JAMA 2001;286:2568- Sterling et al (JHU), AIDS 2001;15:2251-2257- Pallela et al (HOPS), Ann Intern Med 2003;138:620-626- Sterling et al (JHU), J Infect Dis 2003;188:1659-1665

• No virologic or immunologic advantage to starting at CD4 >350 vs. 200-350; increased rate of virologic failure when starting at CD4 <200

- Cozzi-Lepri et al (ICONA), AIDS 2001;15:983-990• Virologic responses comparable among groups with CD4 >200;

slower decline to RNA <500 in those with RNA’s >100,000 at baseline- Phillips et al (SHCS, EuroSIDA, Frankfurt), JAMA 2001;286:2560-2567

• Clinical outcome compromised if Rx begun when CD4 <200 or RNA >100,000- Egger et al (13 cohorts, >12,000 persons), Lancet 2002;360:119-129

Prognosis According to CD4 and RNA:ART Cohort Collaboration

Egger M et al: Lancet 2002;360:119-129

Natural History of Untreated HIV-1 Infection

Time in YearsInfection

CD4Cells

1000

800

600

400

200

0

Early Opportunistic Infections

Late Opportunistic Infections

+

1 2 3 4 5 6 7 8 9 10 11 12 13 14

MACS: CD4 Cell Decline by HIV RNA Stratum

0

-10

-20

-30

-40

-50

-60

-70

-80

-90

Mea

n D

ecre

ase

in C

D4+

Cou

nt p

er Y

ear, cells/mm

3

Plasma HIV-1 RNA Concentration, copies/mL

(n=118) (n=250) (n=386) (n=383) (n=394)

<500 501-3000 3001-10 000 10 001-30 000 >30 000

-30.4 -36.3 -42.3

-39.1 -44.8 -50.5

-50.7 -55.2 -59.8 -59.6

-64.8 -70.0

-70.5 -76.5 -82.9

Mellors et al: Ann Intern Med 1997;126:946-954

CD4 and HIV-1 RNA (I)

• Independent predictors of outcome in most studies

• Near-term risk defined by CD4

• Longer-term risk defined by both CD4 and HIV-1 RNA

• Rate of CD4 decline linked to HIV RNA level in untreated persons

CD4 and HIV-1 RNA (II)

• Good but incomplete surrogate markers- For both natural history and treatment effect

• Thresholds are arbitrary- Disease process is a biologic continuum- Gender specificity of HIV RNA in early-mid stage

disease needs to be considered

• Treatment decisions should be individualized- Baseline should be established- Trajectory determined

HIV Resistance: Underlying Concepts

• Genetic variants are continuously produced as a result of high viral turnover and inherent error rate of RT- Mutations at each codon site occur daily

» Survival depends on replication competence and presence of drug or immune selective pressure

- Double mutations in same genome also occur but 3 or more mutations in same genome is a rare event

- Numerous natural polymorphisms exist

Pre-existence of Resistant Mutants

• Viral replication cycles: 109-1010/day• RT error rate: 10-4-10-5/base/cycle• HIV genome: 104 bp• Every point mutation occurs 104-105 times/day

- In drug naïve individuals» Single and double mutants pre-exist

» Triple and quadruple mutants would be predicted to be rare

HIV Resistance: Underlying Concepts

• Implications- Resistance mutations may exist before drug exposure

and may emerge quickly after it is introduced- Drugs which develop high level resistance with a single

mutation are at greatest risk» e.g., 3TC, NNRTI’s (nevirapine, efavirenz)

- Resistance to agents which require multiple mutations will evolve more slowly

- Partially suppressive regimens will inevitably lead to emergence of resistance

- A high ‘genetic barrier’ needs to be set to prevent resistance

» Potent, combination regimens

HIV Drug Resistance: Definitions

• Genotype- Determines phenotype- Major and minor mutations for PIs

• Phenotype- Drug susceptibility

• Virtual phenotype- Result of large relational genotype and phenotype

database

HIV Drug Resistance: Methodologies

• Genotyping- Different platforms

» Dideoxy sequencing» Gene chip» Point mutation assays

• Phenotyping- Recombinant virus assays

• Virtual phenotyping- Informatics

Tenofovir

K

R

65

ZalcitabineK

R

65

D

T

69

L

V74

M

V

184

Lamivudine

E

D

44

I

V

118

M

VI

184

Abacavir

K

R

65

L

V

74

Y

F

115

M

V

184

Didanosine

K

R

65

L

V

74

Zidovudine

M

L

41

D

RN

K

67 70

L

W

210

YF QE

T K

215 219

E

D

44I

V

118

StavudineM

L41

D

RN

K

67 70

L

W

210

YF QE

T K

215 219

E

D

44I

V

118

Mutations Associated with nRTIs/ntRTIs

www.iasusa.org

Multi-nRTIResistance: 151 Complex

A

V

62

V

I

75

F

L

77

Y

F

116

M

Q

151

Multi-nRTIResistance:69 Insertion

Complex

D

N

67

Insert

R

K

69 70

A

V

62

M

L

41

L

W

210

YF QE

T K

215 219

Multi-nRTIResistance:

NAMs

M

L

41

D

RN

K

67 70

L

W

210

YF QE

T K

215 219

I

V

118

E

D

44

Mutations Associated with nRTIs/ntRTIs

www.iasusa.org

Nucleoside Analog Resistance

TAM’s (M41L, D67N, K70R,

L210W, T215F/Y, K219Q/E/N)

M184V K65R

Confer ZDV resistance thru

ZDV-MP excision

Confers 3TC resistance thru

decreased 3TC-TP incorporation

Confers non-ZDV NRTI resistance thru decreased

analog incorporation

Antagonize K65R

Decreases ZDV resistance thru

decreased ZDV-MP excision

Decreases ZDV resistance thru

decreased ZDV-MP excision

O A U

O

N

T A

3'

5'

O

O

P O-O

O

P-OO

O

5'

N+

-N

primer3'-terminalAZTMP

3'

O A U

A

3'

5'

HO

O

P-OO

O

5'

regeneration of primer free 3'-OH

P OH

O-

O

OP

O-

O

O[R]

Mg2+3'

+

OO

T

N

+N

-N

P

O

OHOP

O

O

O-

P

O

O-

O[R]

where R = AMP (ATP-dependent phosphorolysis)

or

R = H (pyrophosphorolysis)

Pyrophosphorolysis

Courtesy M. ParniakMellors, 9th CROI, 2002

Mutations Selected by NNRTIs

Multi-NNRTIResistance:

Accumulationof Mutations

L V Y G M

I A CI SA L

181100 190106 230

Nevirapine

YK

I AM CI ACLHIN

YL V V G

100103 106 108 181 190188

Efavirenz

LN

YK YL V G

I CI SAI

P

H

100103 108 181 190188 225

Delavirdine

K Y P

CN

181103 236

L

Y

L

188

Multi-NNRTIResistance

Y

L

188

K

N

103

M

V

106

M

V

106

M

V

106

www.iasusa.org

Mutations Selected by PIs

FIRV AFTS

Amprenavir 32 46 47 50 54 73 908410

L V M I G I LII

FIRV I VIL S V MLVMV

9077 82 84 887146363010

FI N I IL I AFTS

VT V MDS

L D M M V VA I LN

Nelfinavir

48 9077 82 8473715410

IRV VL I A V MV SVT

L I V VA I LG G

Saquinavir

10 20 32 33 36 46 54 82 847771 90

FIRV I F IL VL I AFTS

VT V MMR I

RitonavirL K V M M I V VA I LL

IRV II I IL V I AFTVT SA V MMR

82 84777371544632 3610 20 24 90

L K VL M M I V VA G I L

Indinavir

FIRV IL VML AFTS

V M

82 84544610 90L M I V I LMulti-PI

Resistance: Accumulationof Mutations

MR V VI F IL VL VT V MI L P S

L K VL M I VA G I LL I I F L

8273 84 9046 54 7147 50 53 6332 3310 20 24Lopinavir/Ritonavir

II

V

A

A

V

Atazanavir 50 84

VL

71

I

M46

L

I54 82

M

L9088

S

N

I

V

32

www.iasusa.org

Mutations in the GP41 Envelope Gene Associated With Resistance to Entry Inhibitors

Enfuvirtide

DS

G

36

V

I

37

AM

V

38

R

Q

39

T

N

42

D

N

43 HR1 Region

Progress in HIV Disease

HIV Pathogenesis

Monitoring Therapy