Table 1.
Advantages and Limitations of Strategies to Quantify and Characterize the Human Immunodeficiency Virus Reservoir
Approach | Advantages | Limitations |
---|---|---|
Total and integrated HIV DNA by quantitative PCR (eg, real-time, digital PCR) | Highly sensitive, able to be performed on tissues and cells; inexpensive; high throughput | The majority of HIV DNA is defective and does not constitute replication-competent virus; variation in measurements across assays and laboratories; PCR inhibitors exist in various biofluids |
Cell-associated HIV RNA by quantitative PCR | Highly sensitive, can be used to detect the entire spectrum of HIV transcript species within cells; inexpensive; high throughput | Usually measured from bulk cell extracts; sensitive to time of sampling and time from sampling to processing; variation in measurements across assays and laboratories; PCR inhibitors exist in various biofluids |
Ultrasensitive measurement of residual plasma HIV RNA | Highly sensitive, may reflect the “active” HIV reservoir | Remains to be determined if replication-competent virus is exclusively characterized |
qVOA | Provides measurement of replication-competent HIV reservoir | Expensive; time consuming; requires large numbers of cells; variation in results across assays and laboratories; primarily used on cells obtained from peripheral blood; challenging to obtain sufficient viable cells from tissues; some genetically intact viruses may not grow in culture |
Inducible measures of HIV reactivation (eg, TILDA) | Provides measure of the percentage or number of cells in which HIV reactivates upon maximal stimulation; relatively high throughput; requires fewer cells than traditional qVOA | Does not provide measures of replication competence as RNA can be generated from defective viral genomes; challenges with downstream isolation and characterization of genomic DNA or mRNA from individual cells |
Viral protein quantification (eg, HIV p24) | Measures virus with sufficient genetic integrity to drive transcription, translation, and downstream processing | May overestimate viral reservoir size as replication-incompetent viruses may still generate protein |
PET-based imaging/nuclear medicine | Has the potential to survey the whole-body HIV reservoir in various tissues and anatomical locations | In development; requires expression of viral protein and may lack sensitivity required to detect latently infected cells or low levels of viral transcriptional and translational activity in antiretroviral-treated individuals; potential for low signal to noise ratios; expensive; involves in vivo radiation exposure |
Single-cell HIV reservoir characterization | Potential to lead to a greater understanding of the genomic and transcriptional differences between actively infected, latently infected and uninfected cells | Commercially available platforms are expensive and lack the throughput to characterize millions of cells that may be required given low frequency of latently infected CD4 T cells in individuals on ART; higher throughput assays still in development |
Measurement of anti-HIV immune responses (indirect marker) | Titer and avidity of HIV antibodies may represent whole-body, tissue-based HIV persistence and be useful in predicting HIV recrudescence following ATI | Heterogeneous responses; larger longitudinal and cross-sectional studies required to rigorously associate immune responses with reservoir size and HIV rebound dynamics |
Abbreviations: ART, antiretroviral therapy; ATI, analytical treatment interruptions; HIV, human immunodeficiency virus; mRNA, messenger RNA; PCR, polymerase chain reaction; PET, positron emission tomography; qVOA, quantitative viral outgrowth assay; TILDA, Tat/Rev-induced limiting dilution assay.