Cell-Associated Viral Burden Provides Evidence of Ongoing Viral Replication in Aviremic HIV-2-Infected Patients

Studied cohorts.We assessed 45 HIV-2⁺ (16 of which were receiving ART) and 27 untreated HIV-1⁺ patients followed at Hospital de Santa Maria in Lisbon, Portugal, as well as 16 HIV-seronegative age-matched controls. None of the patients had ongoing opportunistic infections or tumors. Tables 1 and 2 describe untreated and treated cohorts, respectively. Although HIV-2-infected cohorts included an increased number of women, non-Caucasian, and elderly individuals, in the current study these factors were not found to significantly impact the parameters under analysis (data not shown). All subjects gave informed consent for blood sampling and processing. The study was approved by the Ethical Board of the Faculty of Medicine, University of Lisbon.

Cell isolation and flow cytometry.Peripheral blood mononuclear cells (PBMC) were isolated immediately after blood collection and characterized by flow cytometry as previously described (9).

DNA and mRNA extraction.DNA was extracted from 5 × 10⁶ PBMC using the QIAamp DNA minikit. For mRNA extraction, 5 × 10⁶ PBMC were lysed (RLT), homogenized (QIAshredder columns), extracted (Oligotex mRNA direct minikit) (all from Qiagen, Valencia, CA), treated with DNase (DNA-free kit; Ambion, Austin, TX), and immediately converted to cDNA. Samples were quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop technologies, Wilmington, DE).

Viremia and proviral DNA.HIV-1 viremia was quantified by a reverse transcriptase PCR (RT-PCR) assay with a detection threshold of 40 RNA copies/ml (Roche, Basel, Switzerland). HIV-2 viremia was quantified using a previously described in-house-developed assay with a detection threshold of 200 RNA copies/ml (54). Quantification of viremia in ART-treated individuals with levels below 200 RNA copies/ml was repeated using a more sensitive real-time RT-PCR assay with a detection threshold of 40 RNA copies/ml as described previously (20). HIV-1 and HIV-2 total viral DNA (integrated and nonintegrated viral DNA species) was quantified using real-time PCR assays that amplify highly conserved regions in HIV-1 and HIV-2 gag with a detection range of 7 orders of magnitude and a sensitivity of 5 copies as we have previously described (52). Test cutoff values were used to calculate the median and the correlations with other parameters when levels were below the cutoff.

gag and tat mRNA.Eighty ng of mRNA was reverse transcribed using the Superscript II reverse transcriptase kit (Invitrogen, Carlsbad, CA), and 250 nM random hexamers was quantified in duplicate using cDNA (1 μg) in a PCR mixture (50 μl) containing 25 μl Platinum quantitative PCR SuperMix-UDG, 1 μl ROX reference dye (50×), 5 mM MgCl₂ (all from Invitrogen), and variable concentrations of the following primers and probes: HIV-1 gag F2 (10 pmol/μl), 5′-GGGAGAATTAGATCGATGGGAAA-3′; HIV-1 gag R1 (10 pmol/μl), 5′-GCTCCCTGCTTGCCCATA-3′; HIV-1 gag probe, 5′-6-carboxyfluorescein (FAM)-CCCTGGCCTTAACCGAATT-minor groove binder (MGB)-3′; HIV-2 gag F2 (10 pmol/μl), 5′-CGCGAGAAACTCCGTCTTG-3′; HIV-2 gag R2 (10 pmol/μl), 5′-CACACAATATGTTTTAGCCTGTACTTTTT; and probe HIV-2-gag, 5′-FAM-CCGGGCCGTAACCT-MGB-3′. tat multiply spliced (MS) mRNA expression was quantified using the following primers and probes: HIV-1 tat F2.3 (6 pmol/μl), 5′-GACGAAGAGCTCCTCAAGACA-3′; HIV-1 tat R2.4 (6 pmol/μl), 5′-GAGACAGAGACAGATCCGGTC-3′; HIV-1 tat probe, 5′-FAM-TCTCTATCAAAGCAACCCGC-MGB-3′; HIV-2 tat F3.5 (10 pmol/μl), 5′-AGGGGCTCGGGATATGTT-3′; HIV-2 tat R3.1 (10 pmol/μl), 5′-TCTGTATCCACCGTCGTTTC-3′; and HIV-2 tat probe, 5′-FAM-TGCATCAGACAAATC-MGB-3′, in an ABI Prism 7000 SDS (Applied Biosystems, Foster City, CA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control (FAM/MGB probe, nonprimer limited; Applied Biosystems; standard curve with cDNA generated from pooled PBMC from 3 seronegatives). Standard curves for tat were generated using amplifications of synthetic oligonucleotides (GenScript Corporation, Piscataway, NJ) corresponding to 200-bp fragments of HIV-1 and HIV-2 tat transcripts, and those for gag were as described previously (52). The median values and the correlations with other parameters were calculated using the tat- and gag-relative quantification values of patients presenting detectable levels of the transcripts.

HIV-2 sequencing.The in-house methodology was used to sequence a 1,280-bp HIV-2 pol gene fragment, including the entire protease and part of the reverse transcriptase from plasma samples. Viral RNA was retrotranscribed and amplified using the Access RT-PCR core reagents kit (Promega, Madison, WI) and outer primers JA218 ([+1859] 5′-GAA AGA AGC CCC GCA ACT TCC-3′) and JA221 ([−3258] 5′-GCT CTG CTT CTG CTA ATT CTG TCC A-3′) as described previously (54). A nested PCR was performed; a second amplification was carried out using AmpliTaq Gold PCR master mix and the inner primers JA219 [(+1898) 5′-AGG GGC T(A/G)A CAC CAA CAG CAC-3′] and JA220_MOD [(−3178) 5′-GTC TTT ATI CCT GGG TAG AI(T/G) TGT G-3′]. Cycle sequencing was accomplished with the BigDye Terminator version 3.1 (v3.1) cycle sequencing kit according to the manufacturer's recommendations and using four sequencing primers: JA219, JA220 [(−3178) 5′-GTC TTT AT(T/C) CCT GGG TAG ATT TGT G-3′], JA222 ([+2525]) 5′-ACC TCC AAC TAA TCC TTA TAA TAC C-3′), and JA223 ([−2625] 5′-ACT GAA TTT CTG TGA AAT CTT GAG T-3′). Purified products were run on an ABI Prism 3100 genetic analyzer according to the same protocol but were adjusted to HIV-2-specific settings; nucleotide sequences were analyzed with SeqScape software version 2.5 (all from Applied Biosystems) by alignment with the ROD HIV-2 reference strain (GenBank accession number M15390).

Statistical analysis.Statistical analysis was performed using GraphPad Prism version 5.00 (GraphPad Software, Inc., SD) using the Mann-Whitney test, Spearman's coefficient, and Fisher's exact test. P values of <0.05 were considered significant. The test cutoff value was used to calculate the median if levels were below the cutoff.

Ongoing viral replication in untreated HIV-2 infection.In order to investigate the degree of ongoing viral replication during the natural history of HIV-2 and HIV-1 infections, we compared untreated HIV-2 and HIV-1 cohorts matched for the degree of CD4 T cell depletion (Table 1), although length of infection was likely greater in HIV-2⁺ than in HIV-1⁺ individuals. As expected, viremia was significantly lower in HIV-2 than in HIV-1 infection (3, 5, 46, 50, 54). Twenty-two out of 29 HIV-2⁺ patients had levels below the test cutoff (aviremic), and the highest viremia was 2.6 × 10⁴ RNA copies/ml (Table 1). Conversely, the proviral DNA loads, as previously reported, were similar (6, 24, 45, 52), suggesting comparable numbers of infected cells in these two groups despite their distinct viremias (Table 1). Of note, although no significant correlation was found between proviral DNA and viremia in HIV-2 infection, significantly higher proviral levels were found in viremic than in “aviremic” individuals (P = 0.0396).

tat mRNA, a multiply spliced (MS) HIV transcript, is thought to be expressed mainly in recently infected cells and/or cells actively producing virus (31, 32, 38, 53). We found a significantly higher number of individuals with undetectable tat mRNA in the HIV-2 than in the HIV-1 cohort (P = 0.0014; Fisher's exact test). Moreover, HIV-2⁺ patients with detectable tat mRNA featured significantly lower levels than their HIV-1+ counterparts (Fig. 1A), and a significant correlation with viremia was observed only with the HIV-1 cohort (r = 0.4791, P = 0.0443, n = 19 for the HIV-1 cohort; r = 0.0312, P = 0.9349, n = 8 for the HIV-2 cohort). Although no correlation with CD4 T cell levels were observed (Table 3), individuals with low CD4 T cell counts tended to have higher levels of tat mRNA (see Fig. 1 posted at http://www.imm.fm.ul.pt/dl/SoaresEtAlSupplementalFigure1AndLegend.pdf).

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FIG. 1.

tat and gag mRNA expression and proviral DNA levels in untreated HIV-2 and HIV-1 infections. Graphs show the expression of tat (A) and gag (B) (as arbitrary units) relative to GAPDH in those individuals with detectable levels of these transcripts, from a total of 25 HIV-2⁺ and 23 HIV-1⁺ individuals evaluated. Open symbols refer to patients with viremia below the cutoff of the test, and closed symbols to patients with detectable viremia. Proviral DNA levels were compared for untreated HIV-2⁺ (25 of 25) and HIV-1⁺ (20 of 23) individuals with detectable and undetectable tat (C) and gag (D) mRNA transcripts. Bars represent medians.

Conversely, gag mRNA, an unspliced (US) HIV transcript, was similarly expressed in the HIV-1 and HIV-2 cohorts (Fig. 1B), which included an equal number of individuals with undetectable expression levels. Of note, no correlation with viremia was found for either infection.

Furthermore, on comparing viremic and aviremic HIV-2⁺ individuals, we observed no significant differences between the number of patients with detectable tat and gag mRNA transcripts, and we found similar levels of expression of tat and gag mRNA (Fig. 1A and B), although these results should be interpreted cautiously, given the small number of individuals assessed.

A low ratio of gag to tat mRNA levels has been suggested as a marker of active viral transcription and of HIV-1 disease progression (22, 37, 49). We found a significantly higher gag-to-tat ratio in HIV-2⁺ than in HIV-1⁺ patients (P = 0.0118), although no association with viremia was found in either cohort.

Of note, neither tat nor gag mRNA expression levels nor the gag-to-tat ratio significantly correlated with the proviral DNA load in either HIV cohort. Moreover, when we subdivided the HIV-1 and HIV-2 cohorts on the basis of detectable and undetectable viral mRNA, no differences in proviral DNA levels were observed (Fig. 1C and D). Thus, no direct relationship appears to exist between the number of infected cells, as estimated by proviral DNA, and the levels of tat and gag transcription in PBMC during untreated HIV-2 and HIV-1 infections, suggesting a significant contribution of archived viral DNA.

Overall, HIV-2⁺ individuals exhibited reduced levels of tat and similar levels of gag transcripts compared to their HIV-1⁺ counterparts, translating into a higher gag-to-tat mRNA ratio in the former.

Relationship of plasma and cell-associated viral load with CD4 T cell depletion and T cell activation in HIV-2 infection.We have previously shown that T cell activation markers were similarly upregulated in HIV-1 and HIV-2 infections when patients were matched for CD4 T cell levels, suggesting that CD4 T cell depletion is more directly linked to immune activation than to viral load (28, 55). Here, we assessed the relationship between cell-associated viral mRNA and DNA and the hyperimmune activation observed for HIV-2 and HIV-1 infections.

With respect to gag mRNA expression, we found a direct correlation with the levels of HLA-DR expression (Table 3) and HLA-DR and CD38 coexpression (r = 0.5150; P = 0.0287) within CD4 T cells in HIV-2⁺ individuals; this correlation did not reach statistical significance in HIV-1⁺ individuals. No correlations were found with CD8 T cell activation levels (Table 3).

Conversely, we observed that the levels of tat mRNA in HIV-1⁺ individuals directly correlated with CD8 T cell activation levels, measured either as the proportion of HLA-DR⁺ CD38⁺ (Table 3) or CD38⁺ cells (r = 0.6196; P = 0.0047), as well as CD38 mean fluorescence intensity (MFI; r = 0.7386; P = 0.0003). Clear trends for an association between tat mRNA levels and CD8 T cell activation were also observed with the HIV-2 cohort, but no significant correlations were found with CD4 T cell activation in either infection (Table 3).

Of note, proviral DNA levels did not directly correlate with either CD4 or CD8 T cell activation in HIV-1⁺ and HIV-2⁺ individuals (Table 3), further suggesting that a significant component comprises archived quiescent virus.

In spite of the narrow range of HIV-2 viremia (undetectable to 26,263 copies/ml), significant positive correlations were found between viremia and several T cell activation markers (Table 3) (percentage of HLA-DR⁺ CD38⁺ within CD4 T cells: r = 0.5685, P = 0.0013; percentage and MFI of CD38 within CD8 T cells: r = 0.6196, P = 0.0047, and r = 0.5330, P = 0.0029, respectively). In order to further assess the relative contribution of viremia, we compared viremic and aviremic HIV-2⁺ individuals (Table 1). Although the former showed significantly higher CD4 and CD8 T cell activation than the latter (Fig. 2A and B), both viremic and aviremic HIV-2⁺ individuals had significantly higher levels of T cell activation than seronegatives did (Fig. 2A and B) and exhibited strong negative correlations between CD4 T cell numbers and CD4 and CD8 T cell activation levels (Fig. 2C and D).

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FIG. 2.

Relationship between viremia, absolute CD4 T cell numbers, and T cell activation in untreated HIV-2 infection. Proportion of CD4 T cells expressing HLA-DR (A) and CD8 T cells coexpressing HLA-DR and CD38 (B) in seronegative individuals (white bars) and untreated HIV-2 patients (dark gray bars). HIV-2 patients were further stratified into viremic (black bars) and aviremic groups (light gray bars). Bars represent median ± interquartile range. Correlations between CD4 T cell depletion and proportion of cells expressing HLA-DR within the CD4 T cell subset (C) and proportion of CD8 T cells coexpressing HLA-DR and CD38 (D).

Overall, our data not only demonstrated that T cell activation was strongly associated with CD4 T cell depletion, both in viremic and aviremic HIV-2⁺ individuals, but also supported a contribution, even at low levels, of circulating virus to both CD4 and CD8 T cell activation. Moreover, we showed that gag mRNA was directly related to CD4 T cell activation and tat mRNA to CD8 T cell activation, suggesting an overall impact of viral transcripts upon T cell activation.

Cell-associated viral mRNA and DNA in HIV-2⁺ patients under ART.In order to further dissect the impact of cell-associated viral DNA and RNA upon HIV-2 immunopathogenesis, we assessed these parameters in patients receiving ART (Table 2). This cohort exhibited significantly lower CD4 T cell counts than untreated HIV-2⁺ individuals did (P = 0.0046), in agreement with previous reports showing a limited CD4 T cell recovery in ART-treated HIV-2 infection (1, 19, 29, 40, 51, 56). Viremia levels were similar in the treated and untreated HIV-2 cohorts, due to the low-level viremia detected in some of the ART-treated HIV-2⁺ patients (Table 2).

Of note, in contrast with the progressive decline in proviral DNA levels usually observed with ART-treated HIV-1⁺ patients (23, 58), proviral DNA did not significantly differ between untreated and treated HIV-2⁺ patients (Fig. 3A), despite the prolonged treatment. Moreover, treated and untreated HIV-2 patients featured similar levels of gag mRNA expression, whereas tat expression was significantly higher in the treated cohort, although the reduced number of individuals with detectable tat transcripts preclude a definitive interpretation (Fig. 3A). No significant differences were found between the number of patients with detectable tat and gag mRNA transcripts within treated and untreated groups. These results showed significant amounts of ongoing viral replication in ART-treated HIV-2 infection, suggesting a limited effectiveness of the ART regimens in HIV-2 infection, which justifies further exploration in large cohorts treated with optimized ART regimens.

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FIG. 3.

Impact of ART on HIV-2 plasma and cell-associated viral load, CD4 T cell frequency, and T cell activation. (A) Levels of proviral DNA and tat and gag mRNA (expressed as arbitrary units) were compared in untreated and ART-treated HIV-2⁺ individuals. Open symbols represent patients with viremia below the cutoff of the test, and closed symbols patients with detectable viremia. Bars represent medians. (B) Longitudinal study of a representative ART-treated HIV-2⁺ patient. Day 0 was defined as the therapy initiation date. The vertical dashed lines indicate the periods under specific ART regimens (ABC, abacavir; 3TC, lamivudine; AZT, zidovudine; LPV, lopinavir; RTV, ritonavir; FTC, emtricitabine; TDF, tenofovir). Mutations in protease (PR) and reverse transcriptase (RT) were analyzed before therapy (day −37) and after the first ART regimen (day 108). Mutations [amino acid changes] found before therapy (day −37) were, in PR, Y14H, N40S, N41D, N68G, K69[K, R], and K70R and, in RT, D17[E, D], W24[G, W], K28R, C38[W, C], K43[K, R], K64R, L74V, K82R, D123G, P126Q, H162Y, V167I, K176S, H228D, W235[G, C, W], and Q245E. Mutations found after first therapy (day 108) were, in PR, Y14H, N40S, N41D, N68G, K69R, and K70R and, in RT, K28R, K43[K, R], K64R, W71R, L74I, K82R, T88[T, P], H96[H, P], G99[A, G], D123G, P126[Q, P], I159[I, L], H162Y, V167I, K176S, M184V, I187[I, L], and L209[P, L]. Proportion of CD4 T cells expressing HLA-DR (C) and CD8 T cells coexpressing HLA-DR and CD38 (D) in seronegative individuals (white bars) and ART-treated HIV-2⁺ individuals (dark gray bars). HIV-2⁺ patients were further stratified into viremic (black bars) and aviremic groups (light gray bars). Bars represent median ± interquartile range.

Additionally, neither the amount of gag and tat transcripts nor proviral DNA significantly differed between treated HIV-2 patients with detectable and undetectable viremia (Fig. 3A), emphasizing that the absence of detectable viremia is not a good surrogate marker for cell-associated viral mRNA and/or DNA during HIV-2 infection.

A rapid emergence of mutations potentially associated with drug resistance has been reported for HIV-2⁺ patients under ART (25, 29, 41). We confirmed the presence of mutations in the reverse transcriptase and protease in our ART-treated HIV-2 cohort (Table 4). These data further support the presence of significant amounts of ongoing replication in these ART-treated HIV-2⁺ patients, leading us to speculate about the potential contribution of ART-induced selective pressure upon the virus to the increased tat transcript levels (Fig. 3).

The selection of adequate ART regimens to treat HIV-2 infection is difficult given the lack of clinical trials. There are currently some phenotypic studies that allow the selection of better first-line therapies than those used a few years ago (16, 61), but the knowledge of HIV-2 resistance pathways is still incomplete. Longitudinal data of an individual HIV-2⁺ patient, in whom the initial ART regimen was associated with the rapid emergence of mutations and virological failure, illustrated that switching to another ART combination was associated with viremia suppression and a decline of cell-associated viral burden (Fig. 3B). This was supported by both a loss of measurable tat and gag transcripts and a decrease in the levels of proviral DNA, both of which are indicative of a successful virological response. Nevertheless, this patient's immunological response was limited, with only marginal CD4 T cell recovery and a partial decrease in both CD4 and CD8 T cell immune activation (Fig. 3B), suggesting that factors other than viral replication are contributing to the poor immunological reconstitution. Our findings support a role for persistent immune activation, given that, as shown in Fig. 3C and D, ART-treated HIV-2⁺ individuals exhibited significantly higher levels of T cell activation than seronegatives did, similar levels of CD4 and CD8 T cell activation in comparison with the untreated HIV-2 cohort, and significant negative correlations between absolute CD4 T cell numbers and the percentages of HLA-DR⁺ within CD4 T cells (r = −0.5147; P = 0.0413) and HLA-DR⁺ CD38⁺ within CD8 T cells (r = −0.5294; P = 0.0350).

Overall, significant amounts of ongoing viral replication were observed with HIV-2-infected patients under ART, highlighting the importance of additional studies of antiretroviral drug efficacy in HIV-2 infection. Furthermore, ART apparently failed to have a significant impact on T cell activation, even in patients with undetectable viremia, possibly contributing to their low CD4 T cell recovery.