hiv cellular pathogenesis iii benhur lee, m.d.. adult v. infant (igg v. iga) ctl response (mhc...
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HIVCellular Pathogenesis III
Benhur Lee, M.D.
Adult v. infant (IgG v. IgA)CTL response (MHC tetramers)p24 antigenimiaAb responseViral load
Viral load “set-point” is a major determinant of disease progression
“Set-point” determined by a balance between the virulence of the viral strain and the quality/strength of host immune response
Control of HIV replication and disease progression by balance of host factors
Viral load “set-point” is a major determinant of disease progression
“Set-point” determined by a balance between the virulence of the viral strain and the quality/strength of host immune response
Viral Load Tests Quantitative (Viral Load determination)
Quantitative RT-PCR (<2x102-1x106) Most sensitive for low levels of viral RNA Requires ~200 l of blood
Branched chain DNA (<5x102-1x106) Most accurate for high levels of viral RNA Requires ~2 ml of blood
NASBA (Nucleic Acid Based Sequence Amplification) (<4x103-1x106)
Clinical interpretation of Viral Load must take into account the type of assay used. Inter-assay differences can differ by as much a 0.5 log.
Quantitative RT-PCR
Quantitative RT-PCR
The “old-fashioned” way
TaqF Q
Taq
Primers and probe anneal to target
Taq begins to displace 5’ end of probe as extension proceeds
Taq
5’ nuclease activity of Taq cleaves off5’ fluorophore on probe
Probe begins to fluoresce as it separates fromQuencher, fluorescence builts up as PCR products accumulate
Real-time PCR
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
Branched Chain DNA Assay
Combination anti-viral Rx can reduced viral loads down toundetectable levels (<50 copies /ml)
RT
Pr
RT Inhibitors
Protease Inhibitors
Entry Inhibitors
Synergism
Lo
g V
ira
l Lo
adPhase 1: Exponential DecayPhase 2: Linear DecayPhase 3: t1/2 of this phase can be used to approximate treatment time for eradication
} Latently Infected Cells--turnover is very slow--relatively resistant to anti-viral Rx
CCR5++
CCR5+
Activation StepIs critical for recovery of virusfrom latent reservoir
CCR5-
CCR5++
Isolate highly purified CD4+ Naïve T-cells
CD4+, CD3+, CD25-. CD69-, HLA-DR-
(Activation Markers)
<0.01% of resting T-cells are latently infected
Limiting Dilution
5 x 106 1 x 106 2 x 105 4 x 104 8 x 103
Activation
add PHA, add CD4+ T cells from HIV-negative donor to rescue virus
Detect viral replication on day 7-9, back-calculate IUPM based on lowest dilution from which virus can be rescued
5 x 106 1 x 106 2 x 105 4 x 104 8 x 103
+ + + + -
IUPM
25
+ + - - - 1
+ + + + + >100
+ + + + +
+ + + + -
+ + + - -
+ + - - -
+ - - - -
- - - - -
Time on HAART
Is Eradication Possible?
Rx period>67 years
Mechanism for persistance of latent reservoir Stability reflects basic biology of memory T cells
Long lived immunity (resting T cells) HepC and Measles specificT cells can be detected >20
years after primary infection Half life of memory T cells (>6 months)
Viremia is NOT completely eliminated Undetectable viral load = No viral replication Continual low-level infection of T cells, replenishment of
latent reservoir
How does one determine low level of viral replication below limits of detection?
Eradication of Viral Reservoirs Treatment Intensification--5-drug HAART
“Flushing out” latent virus T cell activation
Structured Treatment Interruptions “Autoimmunization”
North America
Challenges for an AIDS Vaccine
Antibody response Elicitation of Abs towards neutralizing
epitopes (conserved) Oligomeric vs monomeric Env response
CTL response Conserved CTL epitopes Neutralization Escape mutants Sustaining the response (live viral vectors)