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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 posttest 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 Life
Cycle
Tat = transcriptional
activator
Rev = 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 Infection
Typical Progressor
AIDS
Viral Replication
B
months
years
Nonprogressor
months
years
Clinical Latency
CD4 Level
C
Viral Replication
Primary HIV
Infection
AIDS
CD4 Level
CD4 Level
A
Primary HIV
Infection
Viral Replication
Primary HIV
Infection Clinical Latency
Rapid Progressor
?
months
years
Reprinted with permission from Haynes. In: DeVita et al, eds. AIDS: Etiology, Treatment and Prevention.
4th ed. Lippincott-Raven Publishers; 1997:89-99.
Change in HIV RNA (log10)
Phases of Decay Under the
Influence of Potent Antiretroviral Therapy
0
T1/2 = 1 d (productively infected CD4’s)
-1
T1/2 = 2-4 wks (macrophages,
latently infected CD4’s,
release of trapped virions)
-2
2-4
16-24
Time (weeks)
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
Change in HIV RNA (log10)
Phases of Decay Under the
Influence of Potent Antiretroviral Therapy
0
T1/2 = 1 d (productively infected CD4’s)
-1
T1/2 = 2-4 wks (macrophages,
latently infected CD4’s,
release of trapped virions)
-2
2-4
16-24
Time (weeks)
T1/2 = 6-44 mos (resting,
memory CD4’s)
Model of Post-Integration Latency
Resting
naïve
CD4+ T
cell
+Ag
Activated
CD4+ T
cell
Preintegration
Latency
-Ag
-Ag
Resting
memory
CD4+ T
cell
+Ag
Activated
CD4+ T
cell
Siliciano R et al
+Ag
+Ag
Postintegration
Latency
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;286:25602567
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
1000
800
+
CD4
Cells
600
Early Opportunistic Infections
Late Opportunistic Infections
400
200
0
1
Infection
2
3
4
5
6
7
8
9
Time in Years
10 11 12 13 14
MACS: CD4 Cell Decline by HIV RNA Stratum
Mean D ecrease in CD 4+ Count per Year, cells/mm 3
0
-10
-20
-30.4
-30
-36.3
-40
-42.3
-39.1
-44.8
-50.7
-50
-50.5
-55.2
-60
-59.8
-59.6
-64.8
-70
-70.0
-80
-90
-70.5
-76.5
-82.9
(n=118)
<500
(n=250)
(n=386)
(n=383)
(n=394)
501-3000
3001-10 000
10 001-30 000
>30 000
Plasma HIV-1 RN A Concentration, copies/mL
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
Mutations Associated with nRTIs/ntRTIs
M
Zidovudine
E
41 44
L D
Stavudine
M E
41 44
L D
Didanosine
Abacavir
Lamivudine
70
R
D
K
67
N
70
R
74
R
V
T
L
69
D
74
V
L
K
L
Y
M
65
R
74
V
115
F
184
V
65
R
K
T
K
210 215 219
W YF QE
M
184
V
V
118
I
T
210 215 219
W YF QE
V
118
I
65
K
www.iasusa.org
67
N
L
V
118
I
L
E
44
D
Tenofovir
K
K
K
65
R
Zalcitabine
D
M
184
VI
Mutations Associated with nRTIs/ntRTIs
Multi-nRTI
Resistance:
151 Complex
Multi-nRTI
Resistance:
69 Insertion
Complex
A
V F
62
75 77
I L
V
Y
Q
151
M
L
K
M
41
A
62
67 69 70
L
V
N Inser R
T
K
210 215 219
W YF QE
t
M
Multi-nRTI
Resistance:
NAMs
D 
F
116
E
41 44
L D
www.iasusa.org
D
K
67
N
70
R
V
118
I
L
T
K
210 215 219
W YF QE
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
Pyrophosphorolysis
primer3'-terminal
AZTMP
regeneration of
primer free 3'-OH
3'
5'
O
-O
O
P O
-O
P O
O
O
O
Mg2+
O-
[R]
O
P
O
P
A
O
U
A
U
3'
HO
O
OO
3'
5'
OH
-O
P O
O
O
O
T
+
A
A
3'
N
N+
O
-N
[R]
5'
O
P
O-
O
O
P
O-
O
O
P
OH
O
5'
O
where R = AMP (ATP-dependent phosphorolysis)
N
or
R=H
+N
-N
(pyrophosphorolysis)
Courtesy M. Parniak
Mellors, 9th CROI, 2002
T
Mutations Selected by NNRTIs
Multi-NNRTI
Resistance
Multi-NNRTI
Resistance:
Accumulation
of Mutations
Nevirapine
N M
L
V
Y
G
M
100
106
181
190
230
I
A
CI
SA
L
L K V V
100 103 106 108
I N AM I
Y
181
CI
Y
G
188 190
CLH A
K V
103106
Y
Y
P
181
188
236
N M
C
L
L
L K V V
100 103 106 108
Y
Y
I
www.iasusa.org
Y
188
L
Delavirdine
Efavirenz
K V
103106
N M
I
181
CI
G
188 190
L
SA
P
225
H
Mutations Selected by PIs
Multi-PI
Resistance:
Accumulation
of Mutations
Indinavir
M
46
54
FIRV
IL
VML
L
10
K
20
IRV MR
Ritonavir
L
24
V
M
M
I
32
36
46
I
I
I
IL
I
Nelfinavir
Amprenavir
Lopinavir/
Ritonavir
V
I
L
82 84
A
G
V
54
71
73
77
V
VT SA
90
AFT
S
V
M
V
I
L
82 84
90
I
AFT
V
M
A
V
V
I
L
82 84
L
K
V
L
M
M
10
20
32
33
36
46
54
71
77
I
F
I
IL
VL
VT
I
I
A
G
FIRV MR
Saquinavir
I
L
10
AFT
S
V V
90
V
M
I
L
L
G
10
48
54
71
73
77
82
84
90
IRV
V
VL
VT
S
I
A
V
M
A
V
V
I
N
L
L
D
M
M
10
30
36
46
71
77
82
84
88
90
FI
N
I
IL
VT
I
AFT
S
V
DS
M
L
V
M
I
I
10
32
46
47
FIRV
I
IL
V
M
I
L
K
L
10 20 24
FIRV MR
I
V
I
V
Atazanavir
www.iasusa.org
L
32 33
F
46 47
IL
32
M
46
I
I
V
50
I
54
G
73
84
L
90
V
LVM
S
V
M
I
F
I
L
50 53 54 63
V
L
VL
P
A
G
I
V
L
82 84
90
VT
AFT V
S
M
S
A
V
50
I
54
71
L
L
V
I
I
71 73
N
82
I
84
88
L
90
A
V
S
M
Mutations in the GP41 Envelope Gene Associated
With Resistance to Entry Inhibitors
Enfuvirtide
Q
N N
36 37 38 39
42 43
D
S
T
G
I
V
V
A
M
R
D
HR1 Region
Progress in HIV Disease
HIV Pathogenesis
Monitoring
Therapy