HIV Diagnosis and Pathogenesis Scott M. Hammer, M.D.
Jan 18, 2016
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