1. HIV Cellular Pathogenesis III
Benhur Lee, M.D.

2. Adult v. infant (IgG v. IgA)
CTL response (MHC tetramers)
p24 antigenimia
Ab response
Viral load

3. 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

4. Control of HIV replication and disease progression by balance of host factors

5. 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

8. Viral Load Tests Quantitative (Viral Load determination)
Quantitative RT-PCR (<2x102-1x106)
Most sensitive for low levels of viral RNA
Requires ~200 ml 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.

9. Quantitative RT-PCR

10. Quantitative RT-PCR
The “old-fashioned” way

11. F Q Real-time PCR Taq Primers and probe anneal to target Taq
Taq begins to displace 5’ end
of probe as extension proceeds
Taq
5’ nuclease activity of Taq cleaves off
5’ fluorophore on probe
Probe begins to fluoresce as it separates
fromQuencher, fluorescence builts up as
PCR products accumulate

13. Branched Chain DNA Assay

14. Combination anti-viral Rx can reduced viral loads down to
undetectable levels (<50 copies /ml) RT Pr

15. Protease Inhibitors
Synergism
Entry
Inhibitors
RT Inhibitors

16. Phase 1: Exponential Decay
Phase 2: Linear Decay
Phase 3: t1/2 of this phase can
be used to approximate
treatment time for
eradication
Log Viral Load

17. }
Latently Infected Cells
--turnover is very slow
--relatively resistant to anti-viral Rx

18. CCR5- CCR5++ CCR5+ CCR5++ Activation Step Is critical for
recovery of virus
from latent reservoir
CCR5++

19. Isolate highly purified CD4+ Naïve T-cells
<0.01% of resting T-cells are latently infected
(Activation Markers)
CD4+, CD3+, CD25-. CD69-, HLA-DR-
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

20. 5 x 106
1 x 106
2 x 105
4 x 104
8 x 103
IUPM + + + + - 25 1 + + - - - + + + + +
>100 + -
Time on HAART

21. Is Eradication Possible?
Rx period>67 years

22. 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?

24. Eradication of Viral Reservoirs
Treatment Intensification--5-drug HAART
“Flushing out” latent virus
T cell activation
Structured Treatment Interruptions
“Autoimmunization”

25. North America

26. 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)