Quantifying HIV Reservoirs
Quantifying accurately HIV reservoirs in patients on antiretroviral therapy with the long-term goal of eliminating them as emerged as a major issue in the search towards a cure. However, currently available assays lack the dynamic range needed to document eradication. In order to avoid including analytical treatment interruption as final readout in eradication trials, it is needed to develop more precise biological tools to assess HIV reservoirs, not only in blood but also in tissues and sanctuary sites.
Despite effective antiretroviral therapy (ART), HIV persists in viral reservoirs, in particular in a pool of latently resting CD4+ T cells. There are also other potential cell reservoirs, like macrophages and hematopoietic progenitor cells, precluding HIV eradication.
As research is moving towards potential new strategies for an HIV cure, it is important to define which biological tools will be included in eradication trials. Several groups have published about this in the last few months. We here propose a review of what is available and what is missing. Independently, we remind that the search for new biological tools to assess the HIV reservoir is the subject of a working sub-group in the context of the strategy “Towards a Cure” developed by the International AIDS Society (IAS).
Single-Copy HIV RNA Detection
The single copy assay (SCA) was designed to detect as few as one copy of HIV-1 RNA in 7.5 mL of plasma. Following the advent of ART, plasma HIV-1 RNA was initially shown to undergo biphasic decay kinetics.
The first phase of decay has a half-life of approximately 1.5 days and represents the turnover of free virus and productively infected T cells. The second phase of decay, with a half-life of approximately 28 days, represents the attrition of cells more resistant to HIV cytopathicity, such as partially activated T cells and cells of the monocyte-macrophage lineage. Studies using HIV-1 RNA SCA revealed a third phase of decay (half-life of 39 weeks) that continues below the level of detection of commercial assays, followed by a fourth phase in which viremia does not decay further.
The set point reached by residual viremia correlates with several parameters including the level of pre-ART viremia, the length of time between infection and treatment and the duration of treatment.
Residual viremia is thought to arise from cells that were infected before ART initiation. Such long-lived cells may contribute to this residual very low viremia by the occasional activation of latently infected and/or the continuous or intermittent production of virus by cells resistant to HIV-1 cytopathicity. Whether HIV continues to undergo complete replicative cycles with infection of new cells during effective ART has been the subject of much debate. However, the vast majority of intensification trials showed no effect of a mega-ART regimen on this residual viremia measured by the SCA.
Cell-associated HIV DNA
Quantification of HIV DNA in PBMC has been developed and standardized for more than a decade. However, it is a measure of total HIV DNA in cells and it is possible that the majority of this DNA represents defective viruses in patients on long-term ART.
Within infected cells, HIV DNA can exist as linear nonintegrated forms, circular forms and as an integrated provirus. One popular and widely used technique to quantify the number of cells that contain integrated virus is called Alu-LTR PCR. To improve sensitivity of this assay, many published methods use nested PCR, with the second round of amplification having both primers within the HIV LTR. This increases sensitivity but also the complexity of the assay and multiple replicates and a large number of controls are needed to enhance the accuracy and reproducibility of this method.
Quantification of 2-LTR circles that are episomal forms of nonintegrated HIV DNA containing two copies of the LTR is also a useful tool. 2-LTR circles are produced following infection of a cell and have a relatively short half-life. Therefore, detection of 2-LTR circles is generally considered to be a surrogate marker of recent infection, rather than a marker of the number of residual infected cells.
Cell-associated HIV RNA
The next assay needed to characterize HIV-1 reservoirs is a cell-associated HIV-1 RNA assay with single copy sensitivity. This assay would be useful in investigating the amount of RNA that is produced by infected cells during interventions designed to activate latent virus.
Intracellular HIV RNA assays have however already been established and implemented, but were designed to detect HIV RNA in a population of cells. Methods to detect HIV RNA expression in single infected cells are thus a priority to develop.
Single Genome Sequencing
It is of utility to understand better the genetics of the HIV-1 populations that persist despite antiretroviral therapy. Ultra-deep pyrosequencing allow sthe identification of rare genetic variants and minority drug resistance mutations, which are not detectable by standard genotypic sequencing techniques. The single-genome sequencing assay (SGS) has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination.
A Surrogate Assay for Infectious Virus Recovery
A main problem with HIV DNA quantification is to know whether the measured provirus can potentially, or not, give birth to new infectious viruses. The gold standard for that is a cell culture–based assay. It is a measure the frequency of resting CD4+ T cells carrying latent but replication competent virus. It is based on co-culture of highly purified resting CD4+ T cells from the patient together with
PBMCs from an HIV-negative donor and is measured as infectious units per million cells (IUPM). However this assay is limited in that throughput is low and it is time consuming and resource intensive. In addition, this assay cannot be performed with tissue biopsies.
A relatively high-throughput surrogate for infectious virus recovery would allow for larger scale studies of the impact of therapeutic interventions.
Assessing HIV Activity in Non-blood Compartments
The blood compartment contains approximately 2% of total body lymphocytes. In fact, the gastro-intestinal compartment contains the majprity of CD4+ T cells different mechanisms may contribute to HIV persistence in peripheral blood compared to the gut or other peripheral lymphoid tissues like lymph nodes.
It is also important to evaluate ongoing viral replication in peripheral lymph nodes, compared to PBMC, and which cells are most concerned in tissue reservoirs (like macrophages). Although the brain tissue will never be accessible to routine evaluation, SCA can be applied to CSF. It is also possible to develop non-invasive techniques of brain evaluation during different eradication strategies.
Therefore, it is critical that for new interventions aimed at eradication, quantification of latently and productively infected cells, should be evaluated in tissue as well as blood. Peripheral lymph node biopsies as well as gut biopsies are, probably, important issues for these trials. This will, however, require a standardization of the techniques used.
Assessing Antiretrovirals Activity in Tissue Cells
There is a strong possibility that different ARVs do not reach correctly some tissue compartments. Its is difficult to assess correctly ARVs activity in the central nervous system. However, data are still lacking for several drugs regarding their exact diffusion in the CSF. More importantly, we need to standardize the quantification of intra cellular active metabolites of the different ARVs and be able to apply this measure to specific cell subsets isolated from different lymphoid tissues: lymph nodes, gut-associated lymphoid tissue, broncho alveolar macrophages etc...
Recently, a study reported at the "International Workshop on HIV Persistence, Reservoirs and Eradication Strategies" by 3 research groups (Mario Stevenson, Tim Schacher, Courtney Fletcher) tended to show hudge differences between different antiretroviral drugs in lymphoid tissues.
More data are urgently needed on this issue to definitively sort out whether or not there is a complete blockade of HIV replication in lymphoid tissues during ART.
Adapted from Joseph Wong, reference 4.
In conclusion, the design of new clinical trials testing the possibility to obtain a functional or sterilizing HIV cure has prompted the need for new biological tools to measure residual HIV during effective ART. Some of these tools already exist, but need standardization, others remain to be invented. Improvement in “high throughput technologies” may enable assessment of a larger sample of clinical materials
There is a need for assessment on a single cell level by improved imaging techniques/ single cell analysis techniques.
1-Lewin SR, Rouzioux C. HIV cure and eradication: how will we get from the laboratory to effective clinical trials? AIDS 2011; 25(7): 885-97
2- Pace MJ, Agosto L, Erin H. Graf EH, O'Doherty U. HIV reservoirs and latency models. Virology 2011 Mar 15; 411(2): 344-54
3-Hedskog C, Mild M, Jernberg J, Sherwood E, Bratt G, Leitner T, Lundeberg J, Andersson B, Albert J. Dynamics of HIV-1 Quasispecies during Antiviral Treatment Dissected Using Ultra-Deep Pyrosequencing. PLoS One. 2010; 5(7): e11345
4-Wong J. Evaluating residual HIV reservoirs. 6th IAS Conference, Rome, July 2011
5-Hilldorfer BB, Cillo AR, Besson GJ, Bedison MA, Mellors JW. New Tools for Quantifying HIV-1 Reservoirs: Plasma RNA Single Copy Assays and Beyond. Curr HIV/AIDS Rep. 2012 Jan 4. [Epub ahead of print]
Key words: HIV DNA, HIV eradication trials, HIV new tools, HIV reservoir quantification, HIV reservoirs quantitation, quantifying HIV reservoirs