HIV Cellular Pathogenesis III Benhur Lee, M.D.

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

Quantitative RT-PCR

Quantitative RT-PCR The “old-fashioned” way

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

Branched Chain DNA Assay

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

Protease Inhibitors Synergism Entry Inhibitors RT Inhibitors

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

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

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

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

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)