Structure of HIV
80-130 nm retrovirus with Icosahedral capsid symmetry and composed of two copies of positive-sense single-stranded RNA. HIV genome contains nine genes that encode fifteen viral proteins which are synthesised as polyproteins that produce proteins for virion interior, called Gag, group specific antigen; the viral enzymes (Pol, polymerase) or the glycoproteins of the virion env (envelope).
A capsid surrounds the two strands of RNA which associate with various nuclear proteins of the virus and some of the polymerase proteins. A membrane surrounds the capsid studded with Env proteins (gp 120 and 41)
HIV life cycle
- Envelope proteins bind to CD4 on the surface of CD4+ T cells.
- Chemokine co-receptor (CCR5 or CXCR4) binds.
- The virus is dragged to the cell surface and the membranes of the virus and cell fuse, allowing the core of the virus and its contents including ssRNA to be released into the cytoplasm.
- Reverse transcription of viral RNA genome occurs creating proviral dsDNA which enters the nucleus. Integrase integrates the dsDNA into the chromosome of the host cell, allowing the virus to be transcribed to make more viral RNA which are translated to assemble more virions.
- The newly synthesised virions bud away from the cell, killing the cell.
HIV replication: key features
- Rapid
- Error prone reverse transcriptase leads to rapid evolution of multiple quasispecies
- 10 billion particles produced per day
Which cells are infected by HIV?
HIV infects cells that express CD4 and co-receptors:
- CD4+ T lymphocytes - activated T-cells are killed (massive and early depletion in the gastrointestinal tract) but resting CD4+ T cells experience a latent infection.
- Monocytes and macrophages (express CD4, CCR5 and low level CXCR4) - tissue macrophages in brain (glial cells), lung, gut, bone marrow monocyte precursors, lymphoid tissue macrophages. Macrophages are chronically infected and serve as a reservoir
- Dendritic cells - allow HIV entry, but productive infection is rare
- Thymocytes
- CD34+ progenitor cells
Pathogenesis of HIV
Sexually transmitted:
An abrasion in the epithelium allows the virus through the epithelial layer (DCs can also transfer virus into the lumen). Once in the lumen, virus comes in contact with CD4+ T cells and local expansion occurs. In a period of days to weeks, the virus disseminates to the draining lymph nodes, coming into contact with large numbers of CD4+ T cells. 1-2 weeks post infection, local proliferation occurs with peak plasma virus levels and CD4+ memory cell loss.
Cell-mediated immunity to HIV
Foreign Ag is presented by an APC via its MHC II and recognised by CD4+ of a CD4+ T cell, leading to the cell-mediated immune response.
At which points after infection can one expect to find antibody in the circulation
4-8 weeks post transmission.
Evasion of the immune system by HIV
- Sequence variation: lack of recognition (both CTL and antibody)
- Altered antigen presentation - down regulation of MHC class I molecules by Tat, Vpuand Nef;
- Loss of effector cells: clonal exhaustion, loss of CD4 T cell help, replicative senescence
- Latency: particularly in resting T-cells, macrophages and astrocytes. Latently infected T cells are the major barrier to cure of HIV infection by ART. This reservoir of latently infected cells can persist for the lifetime of a person living with HIV.
- Privileged sites of viral replication (lack of immune system penetrance): Brain, testis, gastrointestinal tract
Treatment for HIV
- Fusion/entry inhibitors
- CCR5 antagonists - bind to CCR5 and inhibit virus/co-receptor interaction.
- Reverse transcriptase inhibitors - prevent active reverse transcription
- Integrase inhibitors
- Protease inhibitors - prevent budding.
ART leads to rapid decline in HIV RNA and CD4 recovery. Life expectancy is normal on ART. Individuals with undetectable levels of virus aren’t able to transmit virus to others. ART must be taken for life due to rapid rebound in virus when ART is stopped
Diagnosis of HIV infection (with time periods)
Within a week to 10 days, viral RNA can be detected in the plasma using qPCR.
2-3 weeks after infection, HIV proteins can be detected in the blood using ELISA.
4-6 weeks after infection, Ab for HIV can be detected.
1st Gen HIV diagnosis method: Western blot. Reactive western blot with purified HIV proteins using patient serum:
- Detects HIV-specific antibodies.
- Requires reactivity to at least two bands of proteins
- Not sensitive to early infection
Step 1: 4th Gen HIV test
- Immunoassays for simultaneous detection of HIV antigen and specific antibody.
- Ultrasensitive immunoassays that can detect p24 antigen and HIV-specific antibodies in the same sample (combination ELISA)
- Identifies HIV p-24 antigen, which appears earlier than antibodies. If negative, individual does not have HIV.
Step 2: If first test results are positive, further confirmatory tests are required. HIV-1/HIV-2 antibody differentiation immunoassay distinguishes between HIV-1/HIV-2, which previously used Western blot cannot. If positive, individual has HIV. If negative or indeterminate, a qPCR is used to ensure accurate detection of early infection. A negative result indicates a false positive of the 4th generation test. A positive result indicates acute HIV infection.
Routine HIV monitoring
1st test: CD4 cell counts (flow cytometry/cell counting)
2nd test: plasma viral load qPCR assay to detect level of viral DNA in the blood (ideal result is negative). If virus load >50cps/mL during ART, likely indication of resistance. Laboratory testing must occur.
How to diagnose resistance to ART?
If virus load >50cps/mL during ART, likely indication of resistance.
Phenotypic resistance (quantitative):
- Phenotypic assays assess the concentration of drug required to inhibit virus replication in vitro (usually cell culture). Results usually expressed as “inhibitory concentration 50%” (IC50) or “inhibitory concentration 90%” (IC90). Useful for viruses that grow easily in culture or where mutations generating resistance may be complex.
- Costly and time-consuming
Genotypic resistance (qualitative):
- Indirect measure of resistance - genotypic assays look for mutations which have been established as a cause of reduced susceptibility in phenotypic assays or in patients. Collected sequence is compared to a reference strain to determine which mutations are present. Mutations are then matched to a known database of resistance mutations. Region of viral gene is PCR amplified and sequenced. Especially useful for viruses that are difficult to culture.
- Cheap and rapid.
