hiv cellular pathogenesis iii benhur lee, m.d.. adult v. infant (igg v. iga) ctl response (mhc...

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

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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)