Sustained suppression of HIV-1 replication to levels undetectable in plasma may be achieved through highly active antiretroviral therapy (HAART), using combinations of reverse transcriptase inhibitors and protease inhibitors (PI) . Despite this, replication-competent virus can be recovered from resting, memory CD4 T cells regardless of the duration of viral suppression [2,3]. In addition, cell-associated HIV-1 genomic and multiply spliced (MS) RNA and transient, unintegrated forms of viral DNA have been detected within the peripheral blood mononuclear cells (PBMC) and lymphoid tissue [4–6], indicating continued, active viral replication rather than drug failure [2–4,7]. The time required for complete viral eradication by HAART, initially estimated at 2–3 years , is now thought to be considerably longer, if achievable at all [7,9,10]. Other potential viral reservoirs such as long-lived cells of the macrophage lineage are also likely to assume importance in HAART patients. In this report, we show that replication-competent HIV-1 can be recovered from highly purified monocytes, and provide evidence for recent infection of these cells.
Blood (30 ml) was initially obtained from individuals with less than 500 copies HIV-1 RNA/ml plasma [Quantiplex HIV RNA 2.0 bDNA assay; Bayer Diagnostics (formerly Chiron Corporation), Emeryville, CA, USA] for more than 3 months. Only cells isolated from five patients with less than 50 copies/ml by the more sensitive 3.0 assay at the time of sampling were used in this study (patients A–J, Table 1). After some success at virus isolation from these patients, another five with less than 50 copies/ml for at least 3 months were recruited (patients L–P, Table 1). Monocyte-enriched populations were isolated using plastic adherence, as previously described . For patients A–J, a second adherence step in 24-well plates was followed by very thorough washing to remove non-adherent cells before the addition of phytohaemagglutinin-stimulated, CD8-depleted PBMC from HIV-negative donors [using Dynal (Oslo, Norway) anti-CD8 magnetic beads according to the manufacturer's protocol]. For patients L–P, the monocyte-enriched populations were further depleted of T cells using anti-CD3 magnetic beads before co-culture. Co-cultures were maintained for 3 weeks without the addition of fresh PBMC. HIV-1 RNA was quantified in culture supernatants using the Amplicor HIV-1 Monitor test (reverse transcriptase–polymerase chain reaction, RT–PCR; Roche Diagnostic Systems Inc., Branchburg, NJ, USA; patients A–J) or the 3.0 bDNA assay (patients L–P). For the detection of drug resistance mutations, RNA was extracted from culture supernatants and the HIV-1 pol gene amplified by RT–PCR and sequenced. The C4 to V4 region of the envelope was also amplified and sequenced. Phylogenetic analysis was performed with reference HIV-1 sequences  using Jukes–Cantor distance matrices and the Fitch/Margoliash tree-building algorithm, and bootstrap values were generated from 100 replicate datasets by consense (ANGIS, Canberra, Australia).
To estimate the level of T cell contamination, messenger RNA was isolated from 1 × 106 purified monocytes using oligo (dT)25 magnetic beads (Dynal), standardized by PCR for β-actin , then used in semi-quantitative PCR specific for T cell receptor mRNA as described previously . All monocyte populations had a 0.1% or less T cell contamination by this method (Fig. 1a). The presence of MS HIV-1 mRNA encoding Nef, Rev, Tat, Vif and Vpr was also determined by semi-nested PCR, as previously described . Proviral DNA was quantified according to the method of Chun et al. , whereas for the detection of circular DNA, extrachromosomal DNA was extracted then amplified with two long-term repeat (2-LTR) circle-specific primers as described previously .
Integrated viral DNA could be detected in all the highly purified monocyte populations (Table 1) at the level of one copy per 103–104 cells (Fig. 1b), as well as in all patient peripheral blood lymphocytes (PBL) preparations tested at similar levels (not shown). The level of proviral DNA found showed no correlation with the period of viral suppression. HIV-1 RNA was detected in co-culture supernatants from five patients (A, B, C, J and L;Table 1); by day 14 in A, B and C but not until day 21 in J and L. Virus could not be detected in any of the cultures within the first 7 days. Again, there was no correlation between virus recovery and the period with undetectable viral load (Table 1). Our results differ from those of Lambotte et al.  who also successfully recovered HIV-1 from three out of five patients after 21 days of co-culture, but only from monocytes activated by either lipopolysaccharide or Staphylococcus aureus. Unlike that group, we were able to recover virus from monocytes without their previous activation. In our hands, the activation of patient monocytes with lipopolysaccharide does not lead to an increase in active replication in these cells (unpublished data). Because HIV-1 has been detected from resting CD4 T cells within 1–7 days of co-culture , our results suggest that fewer monocytes than resting memory CD4 T cells are infected in HAART patients, or that virus is more difficult to recover from the former. Alternatively, virus recovered from monocytes may replicate more slowly. It might be argued that very low numbers of infected T cells could also produce delayed viral replication. As it has been estimated that only 0.2–16.4 per 106 resting CD4 T cells harbour HIV-1 in HAART patients , this is extremely unlikely as the maximum number of purified monocytes co-cultured from any patient was 107 with at most 104 contaminating total T cells.
Reverse transcriptase and protease sequence was obtained for the virus isolates that yielded the highest levels of viral RNA in co-culture supernatants (A, B and C;Table 1). Sequence analysis did not reveal resistance mutations to both classes of antiretroviral drugs, although a number of polymorphisms or compensatory mutations were identified. All three isolates had the L63P substitution in protease, a major natural polymorphism seen pre-treatment [18,19], but also found in indinavir and ritonavir-resistant isolates as a compensatory mutation, improving replicative fitness [19–21]. Isolate C also had the K20R mutation found in some isolates resistant to indinavir, ritonavir or saquinavir, the A71T mutation associated with resistance to indinavir, but also found in PI-naive patients [18,22–24], and the I93L polymorphism found in both untreated and PI-treated patients . Before successful HAART, this patient had been treated with zidovudine and didanosine but never with indinavir. Whereas single amino acid mutations may result in resistance to some PI, phenotypic resistance usually involves the accumulation of multiple substitutions, with three or more generally required before resistance becomes measurable (≥ 4-fold ). Therefore, although isolate C may have some resistance to indinavir, the lack of evidence for resistance to the other PI or reverse transcriptase inhibitors comprising that patient's effective treatment regimen strongly suggests that the isolate recovered was not multiply drug resistant. No additional mutations associated with resistance to any other antiretroviral agents currently or previously used in this or the other patients were found, indicating that the recovery of HIV-1 from monocytes was not caused by treatment failure.
The C4–V4 region of the envelope gene could be amplified only from isolates B and C. Sequences found within the V3 loop were consistent with those expected for non-syncytium-inducing phenotypes, with amino acids at key positions 306 and 320 being similar to those for the non-syncytium-inducing YU-2 rather than the syncytium-inducing NL4-3 reference strain. Also, their V3 sequences contained regions similar to the consensus motif that predicts CCR5 co-receptor usage, S/GXXXGPGXXXXXXXE/D . Viruses recovered from seminal cells of HAART patients have similarly been found to be macrophage tropic , characteristic of strains capable of being sexually transmitted. In contrast, phenotypic analysis of virus produced by resting CD4 T cells from HAART patients showed a syncytium-inducing/CXCR4 phenotype, suggesting that activation of these cells can result in the production of highly cytopathic virus . Our viral sequences were compared with all available published and unpublished sequences, and were found to be unique and to cluster well with subtype B isolates, bearing closest resemblance to North American strains (data not shown).
Circular episomal forms of HIV-1 DNA are labile by-products of replication and are indicative of recent infection . We detected 2-LTR circles in four of the five monocyte populations examined (L, M, N and P), albeit at extremely low levels compared with those found in a control in-vitro-infected PBMC culture (Table 1, Fig. 1c). In the single negative patient (O), they were readily detected in the PBL fraction. In addition, again in four out of five monocyte populations (M, N, O, and P), we were able to detect MS RNA (Table 1, Fig. 1d), providing evidence for active viral transcription in these cells. In three of these, MS RNA was only detected at the highest number of cells tested, whereas patient N monocytes contained levels approaching those in the controls. The presence of MS RNA indicates transcriptional activity, although whether monocyte infection was also translationally active with virion production could not be determined. Although these indicators of ongoing infection have been found in the T cells of HAART patients by a number of groups [4–6,28], their detection in monocytes has not been published previously, only the presence of total viral DNA . Our findings, therefore, suggest recent, low level, active infection in the monocytes as well as the resting, memory CD4 T cells of patients on successful HAART.
Cells of the macrophage lineage have long been considered potential reservoirs of HIV in vivo as a result of their longevity and the minimal cytopathic effect in these cells, with tissue macrophages generally considered to be more important than circulating monocytes [11,29–31]. This study provides evidence, however, that blood monocytes also constitute a continuing source of infectious virus in HAART patients regardless of the length of treatment. The presence of labile forms of HIV-1 DNA and the ability to recover virus from relatively short-lived monocytes (half-life ~3 days) suggest that these cells are newly rather than latently infected, possibly from reservoirs or sanctuary sites such as the central nervous system (CNS). Perivascular macrophages of the brain constitute a major subpopulation of macrophages that are infected with HIV . The CNS is continuously patrolled by monocytes , but the blood–brain barrier prevents the efficient delivery of PI [34,35], the only clinically relevant drugs that can inhibit HIV-1 replication in chronically infected macrophages. The concentrations required, however, are considerably higher than those needed to inhibit the acute infection of T cells [36,37], and so macrophages of the CNS may be able to evade the effects of HIV-specific drugs that are highly effective elsewhere. Improvements in the range and efficacy of antiretroviral therapies and the consideration of additional approaches are required to achieve viral clearance. Whereas the activation-mediated diminution of latent T cell reservoirs of HIV-1 may prove useful , methods that will be effective against monocyte and macrophage reservoirs will also be necessary for eradication to be achievable.
The authors gratefully acknowledge Bayer Diagnostics for the provision of a bDNA assay kit for use in this study. They would also like to thank Christoph Koenigs for technical assistance, John Mills for his critical review of the manuscript and Sharon Lewin for useful discussions.
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