Antiretroviral therapy (ART) suppresses HIV plasma viremia to below the limit of detection in the vast majority of infected individuals. As a result, ART reduces morbidity and mortality in HIV-infected individuals as well as the likelihood of transmitting the virus to uninfected individuals [1–6]. However, it has not been possible to eradicate HIV by ART alone, likely in part due to the persistence of various viral reservoirs in the blood and tissue compartments of infected individuals [7▪▪–9▪▪]. In addition, it has been repeatedly demonstrated that plasma viremia rapidly rebounds in virtually all HIV-infected individuals upon discontinuation of ART regardless of the duration and timing of therapy [10–14]. Consequently, there is a growing interest in developing alternative therapies to eliminate persistent HIV reservoirs and/or to harness host immunity against the virus in order to control viral replication upon discontinuation of ART [15–18]. Currently, suppression of plasma viremia to below the limit of detection by standard assays is a primary goal in the management of HIV infection . Several US Food and Drug Administration (FDA)-approved plasma viral load assays are widely being used to monitor the level of HIV replication and virologic responses to ART in infected individuals . However, other laboratory-based virologic assays are also commonly being used in basic and clinical laboratories to measure HIV burden in clinical specimens obtained from infected individuals, including from those who have achieved maximal suppression of plasma viremia (typically <20–50 copies of HIV RNA/ml) by ART . These laboratory assays are typically PCR-based methods that are designed to quantify the level of cell-associated HIV DNA (total, integrated, and episomal) [22,23] and HIV RNA (unspliced and multiply spliced) . Although these assays provide helpful information with regards to the frequency of cells carrying HIV and the degree and extent of viral replication infected individuals, they cannot distinguish between cells carrying infectious HIV and those harboring defective virus. For this reason, assays capable of detecting replication-competent HIV were developed more than two decades ago and have since been widely used to measure the level of cells carrying infectious virus in HIV-infected individuals [25–27]. This review discusses various methodologies used for detecting replication-competent virus and how such assays are being utilized in basic and clinical research to address the pathogenesis of HIV infection, the persistence of viral reservoirs, and new therapeutic strategies aimed at eradicating the virus in infected individuals receiving ART.
THE INS AND OUTS OF QUANTITATIVE COCULTURE ASSAY
Determining the frequency of cells carrying replication-competent virus in HIV-infected individuals is typically achieved by conducting a quantitative coculture assay [27,28]. This assay is comprised of three major components. First, bulk or subsets of CD4+ T cells are isolated from the peripheral blood mononuclear cells (PBMCs) of infected individuals via blood draw or leukapheresis. Typically, infectious HIV can be detected from a relatively small number of CD4+ T cells (1–2 × 106) in viremic individuals (>50 copies of HIV RNA/ml plasma) or in individuals who have received ART for relatively short periods of time (<1 year). However, a substantially higher number of purified CD4+ T cells are required to detect infectious HIV in individuals who have received ART for more than 5 years and those who initiated therapy during the early/acute phase of infection. Enrichment of bulk or subsets of CD4+ T cells (such as resting CD4+ T cells) is typically achieved by using one of the many commercially available cell enrichment kits that yield high levels of purity (>95–99%). Second, highly enriched CD4+ T cells are accurately enumerated, preferably using an automated cell counter, followed by placing the cells in culture using a limiting dilution approach (five-fold dilutions starting from 1 × 106 cells down to 320 cells per well in duplicate) [26,27] and/or culturing replicates of 5–10 × 106 cells [28,29]. The cell cultures are then maximally stimulated for viral production with irradiated PBMCs from healthy HIV-negative donors in the presence of either phytohaemagglutinin or anti-CD3 antibody for 24 h. In order to further propagate the virus produced by the CD4+ T cells from HIV-infected individuals, anti-CD3-stimulated, CD8-depleted PBMCs from healthy HIV-negative donors are added to the culture on days 1 and 7 followed by periodic removal of cells and replenishment of fresh culture medium containing interleukin (IL)-2. The cultures are typically maintained for 14–21 days. Finally, HIV-positive wells are identified by HIV p24 ELISA and infectious units per million cells are determined by a maximum likelihood method .
MONITORING INFECTIOUS HIV BURDEN IN THE ERA OF ANTIRETROVIRAL THERAPY
The qualitative and quantitative assays for detecting replication-competent HIV were first performed on unfractionated PBMCs without enrichment of CD4+ T cells [25,30]. Given that CD8+ T cells suppress HIV replication , especially in the coculture setting , a more sensitive assay was then developed in which CD4+ T cells from HIV-infected individuals were enriched to a high purity . This modified quantitative coculture assay was subsequently instrumental to identify and characterize latently infected, resting CD4+ T cells in HIV-infected viremic individuals . This study also provided important insight into the virologic aspects of infected CD4+ T cells by demonstrating that the vast majority of resting CD4+ T cells carrying HIV proviral DNA was replication-defective . In addition, the data from this study suggested that the total body viral burden of the latent viral reservoir carrying replication-competent HIV was very small (generally <0.1% of resting CD4+ T cells) and that such infected cells could be long-lived, may not be recognized by the host immune system, and may resist virus-induced cytopathic effects .
Shortly after the introduction of clinically effective ART, several studies that applied mathematical modeling to investigate the kinetics of plasma viremia and to predict the half-lives of the virus in infected individuals following ART generated considerable optimism that HIV could be eradicated in such individuals after 2–3 years of therapy . However, in 1997, three independent studies involving cohorts of HIV-infected individuals receiving effective ART used quantitative coculture assays to demonstrate that the latent viral reservoir carrying replication-competent HIV persisted in virtually all individuals studied, casting serious doubt on the possibility of eradicating virus by ART alone [7▪▪–9▪▪]. Having clearly established that CD4+ T cells carrying infectious HIV persisted in infected individuals receiving ART despite sustained suppression of plasma viremia, many studies since have made use of the quantitative coculture assay to detect and monitor levels of infected CD4+ T cells carrying infectious HIV. Notably, this assay was used in a number of longitudinal studies where the objectives were to determine the half-life of infectious latent viral reservoirs and to project the duration of ART that would be required for eliminating these cells in HIV-infected individuals receiving ART. Two longitudinal studies that primarily focused on a cohort of HIV-infected individuals who began ART during the chronic phase of infection suggested that the half-life of the latent viral reservoir carrying replication-competent virus was greater than 44 months and that even a duration of therapy exceeding 60 years may not be sufficient to completely eradicate HIV [34▪▪,35]. However, two other studies demonstrated that the half-life of the latent viral reservoir was substantially shorter (4–6 months) in HIV-infected individuals who initiated ART during the acute/early phase of infection. These studies projected that at least 7–8 years of continuous therapy would be required to eradicate HIV; however, these estimates were based on the assumptions that ART was capable of completely shutting down viral replication and that no other viral reservoirs existed in infected individuals receiving ART [36,37]. Of note, one of the above studies used a modified version of the quantitative coculture assay (along with a conventional assay) in which replicates of 10 × 106 CD4+ T cells were used to evaluate the infectious HIV burden . Despite sharp contrasts between estimated half-lives of the latent viral reservoir reported in the above studies, the general consensus in the field is that this pool of infected cells is unlikely to be eliminated by ART alone and remains a major impediment to the eradication of the virus in infected individuals receiving ART.
Quantitation of HIV-infected CD4+ T cells carrying replication-competent virus may also be very helpful in delineating the efficacy of therapeutic strategies aimed at eliminating persistent HIV reservoirs and/or obtaining a cure for the virus in infected individuals receiving ART. Over a decade ago, one study demonstrated a marked diminution of the size of the pool of latently infected, resting CD4+ T cells carrying infectious HIV in infected individuals receiving ART and repeated cycles of intermittent IL-2 compared to those who received ART alone . More recently, a number of novel therapeutic strategies aimed at achieving the eradication of HIV in infected individuals receiving clinically effective ART have been suggested and several are currently under investigation [15,16]. Among these is valproic acid (VPA), a histone deacetylase (HDAC) inhibitor that was first tested in vitro and ex vivo as a potential agent for purging the latent HIV reservoir . Subsequently, in an in-vivo study that evaluated the HIV burden at two time points using a quantitative coculture assay, a reduction in the latent viral reservoir was observed in a small number of infected individuals receiving ART and VPA [39▪]. However, a number of follow-up studies demonstrated that VPA did not lower the frequency of resting CD4+ T cells carrying infectious HIV more than ART alone [40–42,43▪▪]. More potent HDAC inhibitors, such as suberoylanilide hydroxamic acid [44▪▪], are currently being investigated in several clinical trials as potential virus-purging agents.
The detection of replication-competent HIV by large-scale (i.e. replicates of >5–10 × 106 CD4+ T cells) quantitative coculture assays could potentially help assess the impact of other cure-related therapeutic strategies. In this regard, a number of anecdotal clinical cases have been recently described in which the burden of HIV DNA was reduced to an undetectable level following chemotherapy and stem cell transplantation [45▪▪,46▪▪]. These cases and their impact in the area of HIV eradiation would greatly benefit from the examination of the persistent HIV reservoirs carrying replication-competent virus by conducing quantitative coculture assays on the largest possible number of purified CD4+ T cells. Such quantitative coculture assays that can probe a much greater proportion of infected CD4+ T cells compared to molecular assays would go a long way to understanding the fate of the persistent HIV reservoirs under conditions that are thought to greatly diminish the HIV burden. In addition, the use of a quantitative coculture assay in cure-related studies provides an important advantage over PCR-based molecular assays in that the vast majority of infected CD4+ T cells carrying HIV proviral DNA are noninfectious and as such these molecular approaches may not adequately evaluate the efficacy of novel therapeutic agents in terms of lowering the viral burden.
ADVANTAGES AND DISADVANTAGES OF QUANTITATIVE MEASUREMENTS OF INFECTIOUS HIV OVER OTHER VIROLOGIC METHODS
The quantitative coculture assay provides a unique opportunity to monitor the frequency of cells that could potentially give rise to infectious HIV in infected individuals. Although highly sensitive PCR-based assays can accurately measure the level of cell-associated viral DNA (total, integrated, and episomal) and cell-associated HIV RNA (spliced and unspliced) in the PBMCs and lymphoid tissues of infected individuals, these methods cannot distinguish between cells carrying replication-competent virus from those harboring replication-defective HIV. Given that less than 1% of infected CD4+ T cells carrying HIV DNA are capable of producing replication-competent virus , it is necessary to utilize a method, such as the quantitative coculture assay, to measure the infectious HIV burden. In addition, the quantitative coculture assay (when configured to evaluate replicates of 5–10 × 106 cells per well) allows the examination of large numbers of cells (i.e. >100 × 106 CD4+ T cells) from infected individuals receiving ART [28,29]. This is especially helpful if a study participant has an exceptionally low level of cell-associated HIV DNA or RNA in CD4+ T cells [47▪] because a typical PCR can only accommodate a small amount of nucleic acid (105–106 cell equivalent) per reaction.
There are also several disadvantages/limitations of the quantitative coculture assay when compared to other virologic assays, including PCR-based molecular assays. First, the reproducibility and sensitivity of the quantitative coculture assay remain to be fully delineated. The confidence intervals for individual measurements involving serially diluted CD4+ T cells (1 × 106 to 320 cells per well in duplicate) are estimated to be relatively wide, at approximately ±0.7 log infectious units per million cells [34▪▪]. On the contrary, most, if not all, PCR-based molecular assays specific for HIV have single copy sensitivity. Of note, laboratory-based plasma viral load assays are now capable of detecting 1–2 copies of HIV RNA per ml of plasma in infected individuals receiving ART [48,49,50▪]. Second, depending on clinical history, the assay may require the purification of large numbers of CD4+ T cells from infected donors [47▪]. It is often necessary to obtain leukapheresis products (>200 × 106 PBMCs) in order to detect infectious HIV in study participants who have been receiving ART for extended periods [51,52]. Such assay requirements increase costs and are labor intensive. Third, the assay detects infectious HIV originating from cells that carry either integrated or unintegrated proviral DNA but cannot distinguish between the two forms and thus is a limitation when trying to establish the source of replication-competent virus. Fourth, the precise association between quantitative coculture assay measurements and other virologic markers remain to be delineated. Finally, the clinical relevance of the level of infected CD4+ T cells carrying infectious HIV remains to be determined in vivo.
Despite more than a decade of intense research and recent therapeutic advances, achieving a true eradication of HIV remains a daunting challenge for the HIV/AIDS scientific community [15–18]. It is estimated that one in 106–109 CD4+ T cells carries replication-competent HIV in infected individuals receiving clinically effective ART [34▪▪,35,47▪,51]. Despite such low frequencies, the presence of persistently infected CD4+ T cells is one of the major impediments to eradicating HIV in infected individuals receiving ART and in whom sustained suppression of plasma viremia has been achieved for prolonged periods [15–18]. Consequently, careful monitoring of the frequency of CD4+ T cells carrying infectious virus is likely to remain an essential element of current and future viral reservoir research efforts. In this regard, a major thrust of current HIV therapeutic research is the development of clinical strategies aimed at eliminating persistent viral reservoirs with the ultimate goal being to eradicate the virus. Therefore, quantitative laboratory assays that precisely measure the number of infected CD4+ T cells carrying replication-competent HIV are likely to play a crucial role in determining the efficacy of therapeutic agents that are designed to either directly target HIV reservoirs or reduce them by enhancing the host immune system. Finally, considerable efforts should be made within the HIV/AIDS scientific community to develop new or improved existing assays for evaluating levels of infectious HIV that are reproducible, clinically relevant, and amenable to high-throughput technologies.
This research was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
Conflicts of interest
The author has declared that no competing interests exist.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
▪ of special interest
▪▪ of outstanding interest
Additional references related to this topic can also be found in the Current World Literature section in this issue (p. 157).
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