DAA-mediated HCV cure reduces HIV DNA levels in HCV/HIV coinfected people

Samaa T Gobran; Amélie Pagliuzza; Omar Khedr; Augustine Fert; Nicolas Chomont; Julie Bruneau; Marina B Klein; Petronela Ancuta; Naglaa H Shoukry

doi:10.1128/jvi.01105-23

. 2023 Dec 5;97(12):e01105-23. doi: 10.1128/jvi.01105-23

DAA-mediated HCV cure reduces HIV DNA levels in HCV/HIV coinfected people

Samaa T Gobran ^1,^2,³, Amélie Pagliuzza ², Omar Khedr ², Augustine Fert ^1,², Nicolas Chomont ^1,², Julie Bruneau ^2,⁴, Marina B Klein ^5,⁶, Petronela Ancuta ^1,^2,^✉, Naglaa H Shoukry ^2,^7,^✉

Editor: Viviana Simon⁸

PMCID: PMC10734513 PMID: 38051044

ABSTRACT

The human immunodeficiency virus (HIV) persists in HIV viral reservoirs despite antiretroviral therapy (ART). Hepatitis C virus (HCV) coinfection is associated with increased HIV reservoir size and residual HIV transcription during ART. Herein, we investigated the impact of direct acting antivirals (DAA)-mediated HCV cure on the size/transcriptional activity of the HIV reservoirs and investigated predictors of HIV reservoirs decline in HCV+/HIV+ coinfected individuals. HCV+/HIV+ (n = 20) and HCV+/HIV− (n = 14) participants were examined prior to DAA treatment (baseline), at the end of treatment (EOT), and at 12–24 weeks after EOT (follow-up). In HCV+/HIV+ individuals, DAA-mediated HCV cure significantly reduced integrated HIV DNA levels, mainly in participants infected with HCV prior to HIV. Integrated HIV DNA, unspliced (US), and multiply spliced (MS) HIV RNA levels were quantified in sorted CD4+ T-cells. Despite the transient elevation of US HIV RNA at EOT, changes in US and MS HIV RNA were not statistically significant. Plasma inflammation markers were measured and DAA also reduced plasma sCD163 and sCD14. Changes in immunological/virological parameters were analyzed using multivariate random forest analysis where pre-ART peak HIV viremia and HCV viral load predicted integrated HIV DNA and US RNA changes. In conclusion, we demonstrate the beneficial impact of DAA on HIV reservoirs and immune activation, with a fraction of HIV reservoirs being DAA-sensitive in people infected with HCV before HIV. Furthermore, we identify HIV/HCV viremia as the top predictors of DAA-mediated changes in the HIV reservoirs. These findings support the need for early ART and DAA treatment in HIV/HCV coinfections.

IMPORTANCE

Antiretroviral therapy (ART) for human immunodeficiency virus (HIV) can control virus replication and prolong the life of people living with HIV (PLWH). However, the virus remains dormant within immune cells in what is called the HIV reservoir. Furthermore, 2.3 million PLWH are also coinfected with hepatitis C virus (HCV) and are at risk of developing chronic liver disease and cancer. HCV treatment with direct acting antivirals (DAA) can completely cure the infection in more than 95% of treated individuals and improve their long-term health outcomes. In this study, we investigated how HCV treatment and cure affect the HIV reservoir. We demonstrate the beneficial impact of DAA treatment as it reduces the HIV reservoirs in particular in people infected with HCV before HIV. These results support the need for early ART and DAA treatment in HIV/HCV coinfections.

KEYWORDS: direct acting antivirals, HIV viral reservoir, CD4+ T-cells, hepatitis C virus

INTRODUCTION

Hepatitis C virus (HCV) infection has emerged as a challenging co-morbidity in people living with HIV (PLWH). The effects of HCV on accelerating the natural progression of HIV infection by augmentation of CD4+ T-cell apoptosis (1), T-cell exhaustion (2), microbial translocation, hepatic injury, and systemic immune activation (3) are well documented. On the other hand, the HIV reservoir, a significant determinant of HIV pathogenesis, has been less explored in the HCV/HIV coinfected population. After HIV DNA integration in the host genome, HIV exploits the cell machinery to generate unspliced (US) viral mRNA transcripts coding for structural proteins (Gag and Pol) and multiply spliced (MS) transcripts giving rise to regulatory proteins such as Tat, a key transcription elongation regulator, and Rev, a protein involved in the nuclear export of US and incompletely spliced HIV mRNA (4, 5). Although antiretroviral therapy (ART) is successful in suppressing HIV plasma viral load to undetectable levels, it fails to eradicate HIV reservoirs, particularly in lymphoid tissues, where ART concentrations may be suboptimal (6, 7). Two studies have reported higher integrated HIV DNA in HCV/HIV coinfected patients compared to HIV-mono-infected ART-treated PLWH (8, 9). This larger HIV reservoir size in HCV/HIV coinfected individuals induced greater expression of MS HIV RNA transcripts (10). In addition, HCV/HIV coinfection is associated with high levels of chronic inflammation and T-cell activation (11), which contributes to the emergence of serious non-AIDS-defining co-morbidities such as cardiovascular, renal, neurologic, and bone diseases (12). Despite the fact that ART diminishes the levels of cellular and soluble activation markers, it fails to fully normalize immune activation and inflammation (13).

In contrast to HIV infection, which requires ART for life (14), HCV cure experienced a major breakthrough in the past decade since the advent of direct acting antivirals (DAA), with >95% cure rates even in people with advanced cirrhosis (15) and HIV coinfection (16). In HCV/HIV coinfected individuals, DAA-induced HCV clearance diminishes immune activation (17, 18), liver disease progression (19), and inflammatory cytokines/chemokines production (18, 20, 21). So far, a few longitudinal studies investigating the evolution of integrated HIV DNA following DAA-mediated HCV cure were performed in ART-treated PLWH and have yielded contradictory results (22). While some studies showed no change (20, 21, 23, 24), another study reported a reduction in the proviral DNA in PLWH undergoing DAA therapy (25). To date, data on predictors of DAA-induced HIV reservoir dynamics remain sparse.

Here, we aimed to characterize the impact of DAA-mediated HCV cure on the persistence and transcriptional activity of HIV reservoirs. We observed a significant reduction in integrated HIV DNA but not HIV RNA in people undergoing DAA treatment. This occurred primarily in individuals infected with “HCV first,” indicative of the existence of a fraction of CD4+ T-cells carrying HIV reservoirs that are DAA-sensitive. Peak HIV viremia and baseline HCV viral loads predicted the decline in HIV DNA and HIV reservoirs transcription activity, respectively.

MATERIALS AND METHODS

Study participants

This longitudinal study was conducted on 20 ART-treated HCV/HIV coinfected patients from the Canadian Co-infection Cohort, undergoing DAA therapy to treat HCV infection. Peripheral blood mononuclear cells (PBMCs) and plasma samples were collected at three-time points: prior to DAA treatment initiation (baseline), after 12 weeks of DAA treatment (EOT), and 12–24 weeks post-treatment (follow-up). At the same three time points, plasma samples were also collected from 14 HCV mono-infected individuals among people who inject drugs from the Montreal Hepatitis C cohort. All participants have provided informed consent for biobanking their samples for research. The study protocol was approved by the Institutional Ethics Committee of the Centre de recherche du CHUM (IRB No. MP-02-2021-9133, SL05.014). All experiments were performed in accordance with relevant guidelines and regulations.

Isolation of total CD4+ T-cells

Total CD4+ T-cells were isolated from 10 million PBMCs by negative selection using negative magnetic beads, as per the manufacturer’s instructions (Miltenyi Biotec, Canada). A cocktail of monoclonal antibodies against CD8a, CD14, CD15, CD16, CD19, CD36, CD56, CD123, TCR γ/δ, and CD235a was used to negatively select CD4+ T-cells. The purity of the CD4+ T-cells was assessed by Flow Cytometry and was >90% for all samples.

RNA/DNA dual extraction

Total DNA and RNA were simultaneously extracted from purified CD4+ T-cells using an Allprep DNA/RNA/miRNA Universal kit (Qiagen), following the manufacturer’s instructions. The quantity and quality (260 nm/280 nm ratio) of the collected DNA/RNA were verified by spectrophotometry (Nanodrop, Applied Biosystems).

Quantification of integrated HIV DNA

Integrated HIV DNA levels were quantified by quantitative real-time nested PCR (10⁵ cells/test in triplicate; detection limit, three HIV DNA copies per reaction), as we previously reported (26, 27). Briefly, a preamplification reaction was performed using external primers specific for the integrated HIV DNA (ULF, Alu1, and Alu2) and human CD3 gene (HCD3OUT5′ and HCD3OUT3′). The amplified products from the first PCR (ProFlex PCR System 9700; Applied Biosystems) were used as templates in a second real-time PCR amplification (RotorGene instrument, Qiagen) with the Rotor-Gene probe master mix (Qiagen) and appropriate sets of internal primers (Lambda (λ) T and UR2 for integrated HIV DNA, HCD3IN5′ and HCD3IN′ for CD3). The UHIV FamZen and CD3 FamZen probes were used to quantify integrated HIV DNA and CD3, respectively. The ACH2 cells carrying one copy of integrated HIV DNA per cell (NIH AIDS Reagent Program) were used for the standard curve.

Quantification of HIV RNA

Nested real-time PCR was performed to quantify US LTR-gag HIV transcripts, as well as MS Tat/Rev transcripts, as we previously described (27). The external primers for the first amplification were (ULF1 and UR1) and (Tat1.4 and Rev) for US and MS RNA, respectively. A second PCR was performed using the internal primers specific for the US (Lamda T and UR2) or MS RNA (Tat2 and Rev). UHIV FamZen and msHIV FamZen probes were used to quantify US and MS RNA, respectively. The first amplification was performed on (ProFlex PCR System 9700; Applied Biosystems), while the second amplification was done on the Rotor-Gene instrument (Qiagen). LTR-gag and Tat-Rev HIV RNA standards were generated from pIDT-Blue plasmid by in vitro transcription (MEGAscriptTM T7 Transcription Kit, ThermoFisher). Primers and probes are listed in Table S1.

Plasma biomarkers measurements

The plasma levels of soluble CD163 (sCD163), soluble CD14 (sCD14), Interferon gamma-induced protein 10 (IP-10), intestinal fatty acid-binding protein (I-FABP) and lipopolysaccharide-binding protein (LBP) were quantified by ELISA (R&D Systems).

Statistical analyses

Statistical analyses were performed using the GraphPad Prism 9 Software (GraphPad Software, Inc.). Non-parametric tests were used to compare groups (Wilcoxon test, Friedman test, Dunn’s multiple comparisons for intra-groups longitudinal comparisons, Mann–Whitney test for inter-groups cross-sectional comparisons). Pairwise correlations were performed using Spearman’s method. All P values were two-tailed and were considered significant only when lower than 0.05. Finally, multivariate random forest regression models were adjusted in R to predict delta (follow-up minus baseline) in HIV DNA, US, and MS RNA. For the adjustment, as well as optimization of the model hyperparameters, the libraries ranger and caret were used in conjunction. The aforementioned optimization was done using threefold repeated (n = 5) cross-validation, in order to overcome the small sample size. For assessment and visualization of variable importance, the packages randomForest, rfPermute, and ggplot2 were used. All statistical analyses were conducted using the R programming language, version 4.2.2.

A detailed description of the methods is available in the Supplemental material.

RESULTS

Characteristics of study participants

A total of n = 20 HCV/HIV coinfected (HCV+/HIV+) and n = 14 HCV mono-infected (HCV+/HIV−) individuals were enrolled in this study. Participant demographics and clinical characteristics are summarized in Table 1 and Table S2. Of note, these two groups differed in age and time since HCV infection, with the HCV−/HIV+ subjects being younger and relatively more recently infected with HCV, as compared to the HCV+/HIV+ participants (Table 1). There were no differences in demographic, virologic, or clinical data. HCV+/HIV+ participants were all receiving ART for at least 1 year before DAA initiation. Except for one participant who had an HIV viral load blip at follow-up, all participants had suppressed plasma HIV RNA throughout the study. Participants with known infection history were stratified into “HCV first” (n = 9) and “HIV first” (n = 10) groups based on which virus was encountered first as confirmed by the first positive PCR test for both HCV and HIV. These two groups were similar in age, sex, HCV status (duration of infection, plasma viral load, and genotype), and the duration of ART, but the “HIV first” group was infected with HIV for longer periods of time compared to the “HCV first” (Table S3). In addition, these two groups were similar in terms of immunological and hepatic function markers (Table S4). All study participants received 12 weeks of DAA therapy for HCV and achieved sustained virologic response (SVR) as confirmed with undetectable plasma HCV RNA 12 weeks after treatment by a validated diagnostic test. Table 2 summarizes the impact of DAA treatment on the immunological and hepatic function markers in the HCV+/HIV+ individuals. DAA did not induce significant changes in median CD4 and CD8 counts, nor the CD4:CD8 ratios, at the end of treatment (EOT) and follow-up compared to baseline (Table 2). However, important reductions in liver stiffness (as determined by Fibroscan), the normalization of liver enzymes (ALT and AST), and an improvement of albumin levels were observed after DAA-mediated HCV cure, as reflected by differences in values between baseline and EOT or follow-up.

TABLE 1.

Socio-demographic and virologic characteristics of study participants

Characteristic	HCV+/HIV+ (n = 20)	HCV+/HIV− (n = 14)
Age (years)^a	53 (36–66)	44 (28–61)^b^,c
Sex (males/females/trans-gender)	(14/5/1)	(13/1/0)
HCV infection
Duration of HCV infection (years)^a	15 (4–26)	3 (1–13)^b^,c
HCV viral load (IU/mL)^a	2,529,043 (122,023–31,248,890)	6,380,734 (19,800–10,611,043)
HCV genotype (1a/1b/3a/4a)	16/2/1/1	10/0/4/0
HIV infection
Duration of HIV infection (years)^a	15 (1–26)	–
Duration of ART (years)^a	9 (1–26)	–
Sequence of infections
Prior onset of HCV infection (HCV first)	9	–
Prior onset of HIV infection (HIV first)	10	–
Unknown sequence of infection	1	–

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^{^a}

Median (range).

^{^b}

Mann Whitney test.

^{^c}

Difference between groups is significant (P value < 0.05).

TABLE 2.

Clinical characteristics of HCV+/HIV+ individuals (n = 20)

Parameter	Baseline	EOT	Follow-up
CD4 count (cells/uL)^c	603.5 (298–1,452)	696 (220–1,618)	625 (233–1,463)
CD8 count (cells/uL)^c	694 (255–2,218)	760.5 (242–2,817)	695 (192–2,223)
CD4/CD8 ratio^c	0.75 (0.3–2.1)	0.75 (0.2–1.9)	0.9 (0.3–1.7)
Liver stiffness (kPa)	7.7 (5.3–31.2)	NA	6.4 (3.7–29.9)^a^,d
ALT (U/L)^c	42 (17–128)	15.5 (8–70)^b^,d	15.5 (7–44)^b^,^d
AST (IU/L)^c	39 (21–136)	24 (11–45)^b^,^d	22.5 (10–48)^b^,^d
Albumin (g/dL)^c	39 (32–49)	41 (38–46)	42.5 (36–50)^b^,^d
Platelets (×10³/mm³)^c	192 (82–295)	190.5 (86–343)	178.5 (99–313)
Total bilirubin (ug/dL)^c	8.9 (3–47)	7.1 (4.7–48.2)	8.6 (3–26)

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^{^a}

Wilcoxon test.

^{^b}

Friedman test.

^{^c}

Median (range).

^{^d}

Difference from baseline is significant (P value < 0.05).

DAA-mediated HCV cure reduced integrated HIV DNA levels mainly in patients infected with HCV prior to HIV

Changes in integrated HIV DNA levels in CD4+ T-cells of HCV+/HIV+ individuals (n = 20) were analyzed before (baseline) and after DAA-mediated HCV SVR (EOT and follow-up). Results indicate a statistically significant reduction in integrated HIV DNA levels at follow-up as compared to baseline (approximately 0.62 median fold-decrease; P = 0.003 Fig. 1A; Fig. S1A and B). The decline in HIV reservoirs was mainly driven by changes in the “HCV first” group (P = 0.006) (Fig. 1B). On the other hand, the overall levels of integrated HIV DNA in the “HIV first” group remained stable, with no statistically significant differences between baseline and follow-up (Fig. 1C). Of note, within the “HIV first” group, only three participants failed to reduce proviral HIV DNA levels, two of whom had a trivial rise, while the third had a twofold increase at follow-up (Fig. S1C). Further analysis of the third participant revealed an HIV viral load blip of 25,340 copies/mL and a CD4 count of 241 cells/µL at follow-up. The exclusion of this outlier did not change the overall results (Fig. S1D). No other differences in the clinical or laboratory parameters were found between the study participants that could account for the observed disparity. Remarkably, at the follow-up time point, we detected significantly lower levels of integrated HIV DNA among the “HCV first” versus the “HIV first” group (P = 0.017), despite no statistically significant differences at baseline (Fig. 1D and E). Taken together, these results demonstrate a significant decrease in integrated HIV DNA levels after DAA compared to baseline (DAA-sensitive HIV reservoirs), primarily in participants who encountered HCV prior to HIV.

Fig 1 — DAA-mediated HCV cure reduced integrated HIV DNA levels mainly in patients infected with HCV prior to HIV. (A) Integrated HIV DNA was evaluated in HCV+/HIV+ individuals (n = 20) before DAA treatment initiation (baseline), at the EOT, and 12 to 24 weeks after finalizing DAA therapy (follow-up). Viral DNA copies were calculated relative to 10⁶ CD4+ T-cells. Shown in grey individuals who were infected with “HCV first” (n = 9) and in black individuals infected with “HIV first” as well as one unidentified participant (n = 11). (**B and C**) Integrated HIV DNA in “HCV first” (n = 9) or “HIV first” (n = 10) HCV+/HIV+ individuals. For A, B, and C the statistical comparisons were performed longitudinally using the Friedman test and Dunn’s multiple comparisons and P values are shown. (**D and E**) Box and whisker plots representing levels of integrated HIV DNA at baseline and follow-up time points following DAA in HCV+/HIV+ individuals who were either “HCV first” or “HIV first.” Mann–Whitney test was used for statistical analysis, P value is shown.

The decrease in HIV DNA levels was not associated with a decline in HIV transcription

Out of the 20 subjects studied, US RNA was detectable in 19 subjects [HCV first (n = 8), HIV first (n = 10), and one with unidentified infection history] and MS RNA was detectable in 16 subjects [HCV first (n = 7), HIV first (n = 8), and one with unidentified infection history]. Levels of US HIV RNA did not significantly change at follow-up compared to the baseline, albeit significant variations between EOT and follow-up (P = 0.028) (Fig. 2A). To gain better insight into these dynamics, the fold change in US HIV RNA levels were analyzed. Results indicated a transient elevation in US HIV RNA at EOT in 12/19 individuals (63%), which subsequently declined at follow-up, except for three participants who had a further increase (Fig. S2A). It is noteworthy that significant differences in US HIV RNA levels between EOT and follow-up were only observed in the “HCV first” (n = 8, P = 0.037), but not in the “HIV first” group (n = 10; P = 0.790). This could be explained by the fact that within the “HCV first” group, the increase in US HIV RNA at EOT was transient and decreased at follow-up (Fig. S2A). On the other hand, MS HIV RNA levels (detectable in n = 16 subjects) did not significantly change upon DAA-induced HCV clearance (Fig. 2B), in both “HCV first” (n = 7) and “HIV first” (n = 8) groups (Fig. S2B). We evaluated the transcriptional activity of the HIV reservoir by calculating the ratio between US or MS HIV RNA and integrated HIV DNA, as previously reported (28). No differences were found in both ratios among baseline, EOT, and follow-up (Fig. 2C and D). In conclusion, except for the transient rise in US HIV RNA at EOT, there were no significant changes in transcriptionally active HIV reservoirs between baseline, EOT, and follow-up.

Fig 2 — The decrease in HIV DNA levels was not associated with a decline in HIV transcription. (A) US HIV RNA measured in HCV+/HIV+ individuals (n = 19) at baseline, EOT, and follow-up. (B) MS HIV RNA quantified in HCV+/HIV+ individuals (n = 16) at baseline, at EOT, and, at follow-up. (**C and D**) Ratios between US or MS HIV RNA and integrated HIV DNA were calculated in HCV+/HIV+ individuals at baseline, at EOT, and at follow-up. Shown in grey individuals who were infected with “HCV first” and in black individuals infected with “HIV first” as well as one unidentified participant. The statistical comparisons were performed using the Friedman test and Dunn’s multiple comparisons, P values are shown.

DAA-mediated HCV cure reduced plasma levels of sCD163 and sCD14 in HCV+/HIV+ individuals

To explore other potential benefits of DAA treatment, we quantified levels of different soluble factors linked to inflammation and gut barrier dysfunction such as sCD163, sCD14, LBP, IP-10, and I-FABP. All of which were previously associated with HIV disease progression and the development of non-AIDS co-morbidities(13, 22). The levels of all measured markers were significantly higher in HCV+/HIV+ versus HCV+/HIV− patients at baseline, except for IP-10 (Fig. S3A). In HCV+/HIV+ individuals, sCD163, sCD14, and LBP levels decreased at follow-up post-DAA compared to baseline (P = 0.0428, P = 0.0133, and P = 0.0531, respectively), whereas IP-10 reduction at EOT (P = 0.0103) was not maintained during the follow-up time point (Fig. 3A). However, I-FABP showed no differences. In HCV+/HIV− individuals, apart from IP-10 and sCD163 that declined at follow-up (P = 0.0421 and P = 0.07, respectively), other mediators were minimally altered by HCV clearance (Fig. 3B). Finally, in HCV+/HIV+ group, different inflammatory measurements were correlated with HIV reservoir markers at both EOT and follow-up. sCD163 levels positively correlated with HIV DNA and US HIV RNA at EOT and predicted their levels at follow-up (Fig. S3B). In conclusion, while DAA-induced HCV cure prompted a significant decline in sCD163 and sCD14 among the HCV+/HIV+ group, it mediated modest changes in inflammatory markers levels in the HCV+/HIV− group, except for IP-10. In the HCV+/HIV+ group, sCD163 levels correlated significantly with HIV reservoir dynamics.

Fig 3 — DAA-mediated HCV cure reduced plasma levels of sCD163 and sCD14 in HCV+/HIV+ individuals. Analyses of changes in the plasma concentrations of sCD163, sCD14, LBP, IP-10, and I-FABP between baseline, EOT, and follow-up for HCV+/HIV+ (A), and HCV+/HIV− (B). Statistical comparisons were performed using the Friedman test and Dunn’s multiple comparisons, respective P values are shown.

Identification of predictors of HIV reservoir markers changes following DAA treatment

Finally, we aimed to determine the relative importance of baseline parameters (clinical, plasma soluble factors, HIV DNA, and US and MS HIV RNA) in predicting DAA-mediated changes in HIV reservoirs expressed as delta change (follow-up minus baseline), using a random forest model. Baseline HIV DNA, US and MS-RNA were among the top significant predictors for DAA-mediated changes in HIV DNA, US and MS-RNA at follow-up, respectively (Fig. 4). Interestingly, pre-ART peak HIV viremia (the highest HIV viral load ever recorded before ART initiation) along with HCV viral load at baseline were identified as important contributors to the prediction model for delta HIV DNA and US-RNA, respectively (Fig. 4A through C). Spearman correlation revealed that higher baseline HIV reservoir markers foresee a greater decline after DAA therapy (Fig. S4A, B, and C). Interestingly, participants who benefited the most from DAA in reducing integrated HIV DNA and US HIV RNA were those who had high pre-ART peak HIV viremia and high HCV viral loads at baseline, respectively (Fig. S4A and B). This analysis suggests that pre-ART peak HIV viremia and HCV plasma RNA loads are among the predictors of DAA-mediated decline in HIV reservoirs.

Fig 4 — Identification of predictors of HIV reservoir markers changes following DAA treatment. Multivariate random forest analysis for the prediction of delta integrated HIV DNA (A) delta US HIV RNA (B), and delta MS HIV RNA (C) using relevant clinical parameters, inflammation markers, and HIV-associated factors at baseline as input variables. The predictors are ranked from top to bottom according to the importance of each variable. Variables with significant importance (P values < 0.05) are shown in red. Delta (follow-up minus baseline).

DISCUSSION

HIV reservoirs persist in long-lived memory CD4+ T-cells with various antigenic specificities (29) and this represents a major obstacle to HIV cure. HCV/HIV coinfected individuals exhibit a larger HIV reservoir size than HIV mono-infected people (8, 10), but the effects of HCV cure on HIV reservoir dynamics remain controversial. In this study, we demonstrated that DAA-mediated HCV cure was associated with a significant decrease in the levels of integrated HIV DNA in HCV+/HIV+ individuals. DAA also reduced plasma markers of immune activation (sCD163 and sCD14). Pre-ART peak HIV viremia and HCV viral load predicted integrated HIV DNA and US RNA changes. Altogether, these results demonstrate the beneficial role of DAA-mediated HCV cure on multiple HIV-related virological and immunological parameters.

Following DAA treatment in HCV+/HIV+ individuals on ART, we observed a significant reduction in integrated HIV DNA levels (approximately a reduction to 62%). Earlier reports on HIV persistence following DAA-mediated cure demonstrated steady (17, 20, 21, 23, 24) or declined (25) HIV DNA levels. These contradictory results may be due to different samples used (purified CD4+ T-cells versus total PBMCs), different forms of HIV persistence measured (Gag HIV DNA versus integrated HIV DNA), diverse PCR-based techniques (nested PCR versus droplet digital PCR), varying levels of HIV viremia before enrollment (low versus undetectable), as well as different clinical and demographic characteristics of the participants. The post-DAA decline in integrated HIV DNA observed in our study temporally coincided with a decrease in immune activation markers such as sCD163 and sCD14. sCD163 is a cell surface marker expressed on tissue macrophages, including hepatic Kupffer cells. sCD163 is shed in response to bacterial LPS and is linked to progression to AIDS and all-cause mortality in HIV patients (30). In our analysis, sCD163 correlated with integrated HIV DNA and US HIV RNA at both EOT and follow-up thus underscoring its importance as a predictor of HIV reservoir dynamics. DAA also reduced sCD14, which is a marker of both monocyte activation and microbial translocation (31). Our data is consistent with other studies reporting a rapid decline in the magnitude of several soluble inflammatory mediators as well as activation surface markers (on monocytes and T-cells), concomitant with the reduction in plasma HCV RNA levels (17, 20, 21, 25, 32). These results suggest that DAA-induced clearance of HCV and the subsequent normalization of HCV-driven immune activation and systemic inflammation have a beneficial role and can lead to a reduction in the levels of HIV reservoirs. Interestingly, in our hands the decline in HIV DNA was mainly observed in patients who had HCV prior to HIV infection. Despite a longer duration of HIV infection in the “HIV first” group, this did not translate into a significant increase in integrated HIV DNA compared to the “HCV first” group at baseline (Fig. 1D; Table S3). In addition, there was no correlation between the duration of HIV infection and HIV reservoir markers (integrated HIV DNA, US, and MS HIV RNA) in all HCV+/HIV+ participants at any of the time points studied, regardless of the virus encountered first (data not shown). This is consistent with a recent study showing no decay in latent HIV reservoirs despite a long time of ART-mediated suppression of HIV replication (mean: 22 years); the authors concluded that HIV reservoirs remained roughly stable partially due to the antigen-driven proliferation of CD4+ T-cells (33). Since HIV reservoirs persist in long-lived memory CD4+ T-cells of various antigenic specificities (29), we speculate that prior HCV infection creates a pool of HCV-specific CD4+ T-cells that are permissive to integrative HIV infection. The possibility of clonal contraction of HCV-specific CD4+ T-cells carrying HIV reservoirs after DAA-mediated HCV clearance, thus leading to HIV reservoir decay during ART, remains to be investigated.

We observed no significant differences in the transcriptional activity of HIV reservoirs at follow-up compared to baseline. This is in contrast to two studies reporting significantly elevated US HIV RNA 12 months following DAA (9, 20). Nevertheless, approximately 63% of our study participants exhibited transiently elevated US HIV RNA at EOT. This may be due to the sudden drop in IFN-mediated antiviral immunity (34), as reflected by the normalization of IP-10 in the peripheral blood at EOT. Of note, Meissner et al. demonstrated that liver biopsies of patients who achieved SVR expressed lower levels of type II and III IFNs but higher levels of type I IFNs as compared to pre-treatment (35). Recent in vitro studies demonstrated that once latency is established, type I IFN-α can reverse it by triggering phosphorylation of STAT 5 (36) which binds to the HIV long terminal repeat and activates HIV transcription (37). In addition, ART concentrations may be suboptimal in tissues (6, 7). Thus, the transient peak of US HIV RNA could also be due to the efflux of transcriptionally active cells that were previously compartmentalized to the liver and other lymphoid tissues during chronic HCV infection (38). Indeed, bulk RNA sequencing demonstrated the emergence of CXCR3-expressing T-cells in the peripheral blood 1 to 2 weeks after DAA treatment initiation (39, 40), coinciding with the rapid decline in IP-10 levels (41). Finally, the transient induction of US HIV transcripts at EOT may not be the best indicator of active viral replication as it was not reflected in an increase in plasma RNA levels in our study, as latently infected CD4+ T-cells can initiate transcription that is afterward blocked at the elongation, polyadenylation, or splicing steps (42).

We identified baseline levels of HIV reservoir markers (HIV DNA, US, and MS-RNA) as significant predictors of corresponding changes in HIV reservoirs following DAA. In addition to plasma viral load, HIV DNA and US-RNA were previously shown to be crucial biomarkers in predicting clinical progression following ART discontinuation (43, 44). This is consistent with previous studies demonstrating that higher levels of HIV reservoir markers (HIV DNA and US HIV RNA), while on ART are associated with a shorter time to HIV rebound (43, 44), higher peak of viremia following ART cessation, and failure to reach undetectable viremia on re-starting ART (45). Our results support a model in which DAA treatment reduces the size of HIV reservoirs mainly in people with increased HIV viremia prior to ART and those infected with HCV first, thus accelerating HIV reservoir decay during ART. Furthermore, we demonstrated that individuals with relatively higher plasma HCV RNA at baseline are the ones who benefited the most from DAA treatment. These participants exhibited a preferential post-DAA decline in US HIV RNA, a key indicator of the magnitude and time to viral rebound after early ART interruption and subsequent disease progression in the absence of treatment (46). This multivariate analytical model integrating clinical and HIV reservoirs markers can inform the design of clinical trials to study HIV dynamics in the context of other coinfections and experimental therapeutics.

This study has a few limitations. First, the use of PCR-based techniques for measuring both integrated HIV DNA and HIV RNA. Although broadly used, PCR-based assays overestimate the size of the HIV reservoirs due to the high proportion of defective proviruses in PLWH on ART (47). Further studies using intact proviral DNA assay would be necessary to quantify the integrity of such proviruses (48). Second, we used RT-PCR to quantify MS HIV RNA directly ex vivo in CD4+ T-cells of ART-suppressed individuals, without TCR triggering that likely happens in vivo. Future studies involving in vitro induction assays such as Tat/Rev Induced Limiting Dilution Assay or viral outgrowth assays should be used to precisely measure transcription-competent HIV reservoirs (49). Third, our results may not be representative of the intrahepatic CD4+ T-cells. This point may be addressed in future studies with liver fine needle aspirates. Finally, we could not determine whether DAA treatment reduced overall systemic inflammation by clearing HCV, inducing a decay in the HIV DNA reservoirs, or both. Despite this limitation, our study underscores the beneficial effect of DAA in controlling systemic inflammation, with a potential impact on reducing the risk of non-AIDS co-morbidities.

In conclusion, our study stresses the importance of DAA-induced HCV clearance in reducing levels of integrated HIV DNA and systemic inflammation markers (sCD163, sCD14) in HCV+/HIV+ individuals. This is in line with evidence that HCV cure has proven to be beneficial not only in terms of the liver-related mortality rate and fibrosis regression but also in minimizing HIV disease progression and non-AIDS-related mortality. Of particular importance, our results suggest that a fraction of CD4+ T-cells carrying HIV reservoirs, likely composed of HCV-specific T-cells, is “DAA-sensitive,” mainly in HCV+/HIV+ people who were infected with “HCV first.” Finally, we identified levels of HCV viral load at baseline among predictors of HIV transcription decline following DAA. Future studies are required to evaluate whether HCV-specific CD4+ T-cells harbor a part of the latent HIV reservoirs in ART-treated individuals and whether this reservoir persists upon DAA-mediated HCV clearance, especially in deep tissues such as the liver. The existence of such HIV reservoirs may be a matter of concern for high-risk populations with an increased risk of HCV reinfection following DAA-mediated cure.

ACKNOWLEDGMENTS

This study was funded through grants from the Canadian Institutes of Health Research (CIHR) (PJT-173467 to N.H.S., HOP-120239 and PJT-153052 to P.A.), the National Institutes of Health (NIH) (U19AI159819), Canadian HIV Cure Enterprise Team Grant (CanCURE 1.0) funded by CIHR in partnership with the Canadian Foundation for AIDS Research (CANFAR) and the International AIDS Society (IAS) (CanCURE 1.0; HIG-133050), and the CanCURE 2.0 Team Grant funded by CIHR (HB2-164064) to P.A. Cohorts are partially supported by Fonds de recherche du Québec-Santé; Réseau SIDA/maladies infectieuses. The Canadian Co-Infection Cohort is supported by CIHR (FDN-143270); and the CIHR Canadian HIV Trials Network (CTN222) granted to M.B.K. S.T.G. is supported by a doctoral fellowship from the Canadian Network on Hepatitis C (CanHepC). CanHepC is funded by a joint initiative of the CIHR (NHC142832) and the Public Health Agency of Canada. J.B. and M.B.K. are supported by Tier I Canada Research Chairs.

Contributor Information

Petronela Ancuta, Email: petronela.ancuta@umontreal.ca.

Naglaa H. Shoukry, Email: naglaa.shoukry@umontreal.ca.

Viviana Simon, Icahn School of Medicine at Mount Sinai, New York, New York, USA.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/jvi.01105-23.

Supplemental material. jvi.01105-23-s0001.pdf.

Additional experimental details, Tables S1 to S4, and Fig. S1 to S4.

Click here for additional data file.^{(140KB, pdf)}

DOI: 10.1128/jvi.01105-23.SuF1

ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental material. jvi.01105-23-s0001.pdf.

Additional experimental details, Tables S1 to S4, and Fig. S1 to S4.

Click here for additional data file.^{(140KB, pdf)}

DOI: 10.1128/jvi.01105-23.SuF1

[B1] 1. Körner C, Krämer B, Schulte D, Coenen M, Mauss S, Fätkenheuer G, Oldenburg J, Nattermann J, Rockstroh JK, Spengler U. 2009. Effects of HCV co-infection on apoptosis of CD4+ T-cells in HIV-positive patients. Clin Sci (Lond) 116:861–870. doi: 10.1042/CS20080532 [DOI] [PubMed] [Google Scholar]

[B2] 2. Rallón N, García M, García-Samaniego J, Rodríguez N, Cabello A, Restrepo C, Álvarez B, García R, Górgolas M, Benito JM. 2017. HCV coinfection contributes to HIV pathogenesis by increasing immune exhaustion in CD8 T-cells. PLoS One 12:e0173943. doi: 10.1371/journal.pone.0173943 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B3] 3. Shmagel KV, Saidakova EV, Shmagel NG, Korolevskaya LB, Chereshnev VA, Robinson J, Grivel J-C, Douek DC, Margolis L, Anthony DD, Lederman MM. 2016. Systemic inflammation and liver damage in HIV/hepatitis C virus coinfection. HIV Med 17:581–589. doi: 10.1111/hiv.12357 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4. Rice AP. 2017. The HIV-1 tat protein: mechanism of action and target for HIV-1 cure strategies. Curr Pharm Des 23:4098–4102. doi: 10.2174/1381612823666170704130635 [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

DAA-mediated HCV cure reduces HIV DNA levels in HCV/HIV coinfected people

Samaa T Gobran

Amélie Pagliuzza

Omar Khedr

Augustine Fert

Nicolas Chomont

Julie Bruneau

Marina B Klein

Petronela Ancuta

Naglaa H Shoukry

Roles

ABSTRACT

IMPORTANCE

INTRODUCTION

MATERIALS AND METHODS

Study participants

Isolation of total CD4+ T-cells

RNA/DNA dual extraction

Quantification of integrated HIV DNA

Quantification of HIV RNA

Plasma biomarkers measurements

Statistical analyses

RESULTS

Characteristics of study participants

TABLE 1.

TABLE 2.

DAA-mediated HCV cure reduced integrated HIV DNA levels mainly in patients infected with HCV prior to HIV

Fig 1.

The decrease in HIV DNA levels was not associated with a decline in HIV transcription

Fig 2.

DAA-mediated HCV cure reduced plasma levels of sCD163 and sCD14 in HCV+/HIV+ individuals

Fig 3.

Identification of predictors of HIV reservoir markers changes following DAA treatment

Fig 4.

DISCUSSION

ACKNOWLEDGMENTS

Contributor Information

SUPPLEMENTAL MATERIAL

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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