
==== Front
iScience
iScience
iScience
2589-0042
Elsevier

S2589-0042(24)01895-9
10.1016/j.isci.2024.110670
110670
Article
Metformin facilitates viral reservoir reactivation and their recognition by anti-HIV-1 envelope antibodies
Fert Augustine 12
Richard Jonathan 12
Raymond Marchand Laurence 1
Planas Delphine 12
Routy Jean-Pierre 345
Chomont Nicolas 12
Finzi Andrés 12
Ancuta Petronela petronela.ancuta@umontreal.ca
126∗
1 Centre de recherche du Centre hospitalier de l’Université de Montréal, Montréal, QC H2X 0A9, Canada
2 Département de microbiologie, infectiologie et immunologie, Faculté de médecine, Université de Montréal, Montréal, QC H3C 3J7, Canada
3 Infectious Diseases and Immunity in Global Health Program, Research Institute, McGill University Health Centre, Montréal, QC, Canada
4 Chronic Viral Illness Service, McGill University Health Centre, Montréal, QC, Canada
5 Division of Hematology, McGill University Health Centre, Montreal, QC, Canada
∗ Corresponding author petronela.ancuta@umontreal.ca
6 Lead contact

05 8 2024
20 9 2024
05 8 2024
27 9 11067013 2 2024
27 5 2024
1 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

The mechanistic target of rapamycin (mTOR) positively regulates multiple steps of the HIV-1 replication cycle. We previously reported that a 12-week supplementation of antiretroviral therapy (ART) with metformin, an indirect mTOR inhibitor used in type-2 diabetes treatment, reduced mTOR activation and HIV transcription in colon-infiltrating CD4+ T cells, together with systemic inflammation in nondiabetic people with HIV-1 (PWH). Herein, we investigated the antiviral mechanisms of metformin. In a viral outgrowth assay performed with CD4+ T cells from ART-treated PWH, and upon infection in vitro with replication-competent and VSV-G-pseudotyped HIV-1, metformin decreased virion release, but increased the frequency of productively infected CD4lowHIV-p24+ T cells. These observations coincided with increased BST2/tetherin (HIV release inhibitor) and Bcl-2 (pro-survival factor) expression, and improved recognition of productively infected T cells by HIV-1 envelope antibodies. Thus, metformin exerts pleiotropic effects on post-integration steps of the HIV-1 replication cycle and may be used to accelerate viral reservoir decay in ART-treated PWH.

Graphical abstract

Highlights

• Metformin increases the frequency of productively HIV-infected T cells

• Metformin acts on HIV-1 replication cycle at post-entry and post-integration levels

• Metformin increases BST2 and Bcl-2 expression in productively HIV-infected T cells

• Metformin increases viral reservoir recognition by anti-HIV-Env Abs

Medication; Virology; Molecular microbiology

Subject areas

Medication
Virology
Molecular microbiology
Published: August 5, 2024
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pmcIntroduction

Antiretroviral therapy (ART) efficiently reduces HIV-1 replication to undetectable plasma levels and increases the life quality of people with HIV-1 (PWH).1 However, ART does not eradicate HIV-1 since viral reservoirs (VRs) persist in cells and tissues, a process associated with increased risk of developing non-AIDS comorbidities such as cardiovascular diseases, cancers and metabolic disorders, thus causing accelerated aging (i.e., frailty, dementia).2,3,4,5,6 Those HIV-1 related pathologies are caused by chronic immune activation.7,8 Chronic immune activation in PWH is multifactorial. In fact, suboptimal ART penetration may occur in specific tissues and cell subsets.9 In addition, ART does not restore mucosal CD4+ T cells populations depleted by HIV-1 infection.10 Finally, ART does not block HIV-1 transcription and translation leading to residual HIV-RNA and protein expression.9,11,12 New therapeutic strategies are needed to reduce comorbidities associated with chronic inflammation in PWH. In the absence of an HIV cure, whether strategies targeting HIV transcription and translation could achieve this goal remains to be determined.

The mechanistic target of rapamycin (mTOR) pathway, a key regulator of T cell differentiation and growth via the induction of glycolysis,13,14 was reported to be involved in multiple steps of the HIV-1 replication cycle (i.e., entry, reverse transcription, nuclear transport and transcription).15,16,17,18 Previous work by our group demonstrated that mTOR expression and phosphorylation was preferentially induced in CD4+ T cells expressing the Th17 marker CCR6 upon T cell receptor (TCR) triggering and exposure to the gut-homing modulator retinoic acid. Also, by using a VSV-G-pseudotyped HIV-1 construct, which enters cells via endocytosis independently of CD4 and co-receptors,19 we demonstrated that mTOR activation facilitates the post-entry steps of the HIV-1 replication cycle in RA-treated CCR6+CD4+ T cells.20 Furthermore, we demonstrated that mTOR inhibitors reduce viral outgrowth in CCR6+CD4+ T cells of ART-treated PWH.20 Consistently, Besnard et al., showed that mTOR activation promotes HIV-1 transcription, via mechanisms involving the phosphorylating of CDK9, a subunit of the PTFEb complex, needed for HIV-1 transcription elongation.17 Finally, Taylor et al. showed that mTOR activity is increased in CD4+ T cells from ART-PWH compared to HIV-uninfected individuals; mTOR inhibition in TCR-activated CD4+ T cells leads to a decrease in the pool of dNTPs needed for HIV-1 reverse transcription; and that mTOR activation stabilizes microtubules in HIV-infected T cells to facilitate the nuclear import of HIV-1 pre-integration complexes.18

Knowledge on the importance of mTOR pathway in regulating specific steps of the HIV-1 replication cycle,15,16,17,18,20 prompted mTOR targeting in vivo in ART-treated PWH. In a single-arm clinical trial, 6-month supplementation of ART with everolimus, a direct mTOR inhibitor used as immunosuppressive drug in transplant recipient,21 decreased mTOR activation, as well as the cell-associated (CA) HIV-RNA levels in blood CD4+ T cells,22 thus pointing to a potential direct role of mTOR in modulating HIV transcription in vivo. However, the use of such immunosuppressive drugs is not recommended outside organ transplantation. Metformin, an indirect mTOR inhibitor, is a drug approved by the Food and Drug Administration (FDA) and widely used to treat type-2 diabetes.23 Mechanistically, metformin blocks the first complex of the respiratory chain of the mitochondria, leading to an increase in the AMP/ATP ratio. The change in this ratio leads to an activation of AMP-activated protein kinase pathway, which results in mTOR pathway inhibition.24,25 Our group performed a pilot non-randomized clinical trial in which non-diabetic ART-treated PWH received metformin for 12 weeks, and matched blood and colon biopsies were collected at baseline and the end of treatment for immunological and virological measurements.26,27 Metformin significantly decreased the frequency of colon-infiltrating CD4+ T cells and mTOR phosphorylation in CCR6+CD4+ T cells, and reduced levels of systemic inflammation (i.e., sCD14). Finally, a reduction in HIV-1 transcription, measured as the HIV-RNA/DNA ratio, was observed in CD4+ T cells isolated from the colon in a fraction of study participants (8/13), thus pointing to a potential link between mTOR activation and HIV-1 transcription in T cells carrying VRs.27 Of interest, Guo et al. showed that metformin reduced HIV-1 replication and limited CD4+ T cells depletion in a humanized mouse model reconstituted with primary human CD4+ T cells.28 Another study showed that 24 weeks of metformin administration in complement of ART decreased the frequency of CD4+ T cells with a PD1+TIGIT+TIM3+ phenotype considered as a molecular signature for exhausted cells contributing to HIV persistence.29,30 Whether the metformin supplementation of ART may represent a valuable strategy to decrease immune dysfunction in PWH requires further investigations.

Given the encouraging results of metformin supplementation of ART in PWH,27,31 we sought to identify the steps of the HIV-1 replication cycle modulated by metformin and its mechanisms of action. To this aim, we studied the effects of metformin on HIV-1 replication in CD4+ T cells from HIV-uninfected participants exposed to HIV-1 in vitro. Also, we tested metformin effects on viral outgrowth from CD4+ Tcells of ART-treated PWH. Our results reveal unexpected effects of metformin on various post-entry steps of the viral replication cycle. Metformin increased HIV-p24 expression at single-cell level upon HIV-infection in vitro, without a proportional increase in HIV-DNA integration and viral release, via mechanisms involving increased BST2/tetherin (HIV release inhibitor) and Bcl-2 (pro-survival factor) expression. Also, metformin favored the recognition of reactivated HIV-1 reservoir cells by broadly neutralizing (bNAbs) anti-HIV-Env Abs. Overall, these results support a model in which metformin may be used to accelerate VR decay during ART.

Results

Metformin dose-response testing

To determine the optimal concentration of metformin for experiments in vitro, we first tested the effect of different doses of metformin on the phosphorylation of mTOR and its downstream substrate, the ribosomal S6 (S6). Results demonstrate that metformin at 1 mM efficiently inhibited mTOR and S6 phosphorylation upon TCR triggering, without impacting on cell viability and proliferation, while metformin at 5 mM reduced proliferation and viability (Figure S1). Thus, the dose of 1 mM metformin was considered optimal for CD4+ T cells in vitro. A similar concentration of metformin (1 mM) showed efficacy in another study in blocking the mTOR pathway and HIV-1 replication by reducing oxygen consumption in CD4+ T cells.28

Metformin increases the frequency of HIV-p24+ T cells without a proportional increase in viral release upon infection in vitro

To explore metformin effects on HIV-1 replication, TCR-stimulated CD4+ T cells from HIV-uninfected participants were exposed to a replication-competent CCR5-tropic HIVNL4.3BAL virus upon TCR triggering and culture, in the presence or the absence of metformin (0.5, 1, 5 mM) and INK128 (50 nM), a potent direct mTORC1/mTORC2 inhibitor32 (Figure 1A). Although metformin (1 mM) and INK128 treatment did not significantly impact on levels of HIV-DNA integration at day 3 post-infection (Figure 1B), both reduced HIV-p24 levels in cell-culture supernatant, with metformin showing a dose-response effect (Figure 1C). Cells treated with 5 mM of metformin displayed higher integrated HIV-DNA levels (Figure 1B), however, this concentration reduced cell viability and proliferation (Figures S1E and S1F). Flow cytometry analysis at day 12 post-infection reveals that metformin increased intracellular HIV-p24 expression in T cells with a CD4low phenotype (productively infected) or CD4high (likely bystander cells coated with newly formed virions) (Figure 1D). The HIV accessory proteins Nef and Vpu downregulate CD4,33,34 and their presence in HIVNL4.3BAL explains the downregulation of CD4 on the surface of productively infected T cells. Also, CD4 downregulation precedes HIV-p24 expression,33,34 likely explaining the detection of a CD4lowHIV-p24- population (Figure 1D). It is also noteworthy the atypical appearance of two subsets of CD4lowHIV-p24+ T cells, with medium and high levels of HIV-p24 expression upon exposure to metformin (Figure 1D). In contrast to results in Figure 1C, metformin, at 1 mM dose, increased cell-associated HIV-p24 levels in terms of frequency of CD4lowHIV-p24+ and intensity of HIV-p24 expression (Figures 1E and 1F). Metformin also significantly increased the frequency of CD4highHIV-p24+ T cells, as well as the intensity of HIV-p24 expression (Figures 1G and 1H).Figure 1 Dose-response effects of metformin on HIV-1 replication in memory CD4+ T cells in vitro

(A) Shown is the flow chart for the HIV-1 infection in vitro. Briefly, memory CD4+ T cells from HIV-uninfected donors were stimulated with anti-CD3/CD28 Abs in the absence/presence of metformin (0.5, 1, or 5 mM) or INK128 (50 nM) for 3 days. Then, cells were exposed to the replication-competent NL4.3BaL HIV strain (50 ng HIV-p24/106 cells). Cell-culture supernatants were collected and media containing IL-2 and/or metformin or INK128 was refreshed every 3 days until day 12 post-infection. Shown are statistical analysis for integrated HIV-DNA levels were quantified by real-time nested PCR at day 3 post infection (B) and HIV-1 replication determined as HIV-p24 levels in cell-culture supernatants measured by ELISA at day 9 post-infection (C). Shown is a flow cytometry dot plot analysis of the intracellular HIV-p24 and surface CD4 expression allowing the identification of CD4lowHIV-p24+ cells (productively infected) and CD4highHIV-p24+ cells in one representative donor (D); as well as the statistical analysis of CD4lowHIV-p24+ T cell frequency (E) and their geometric MFI of HIV-p24 expression (F), and of CD4highHIV-p24+ T cell frequency (G) and their geometric MFI of HIV-p24 expression (H). Each symbol represents 1 donor (n = 8; median ± interquartile range). Friedman and uncorrected Dunn’s multiple comparison p-values are indicated on the graphs.

In conclusion, these results demonstrate that metformin increases cell-associated HIV-p24 expression and results in the expansion of HIV-p24+ T cells with CD4low and CD4high phenotypes, likely by promoting HIV-1 cell-to-cell transmission via mechanisms independent of free-virion release.

Effects of metformin on RORC2 expression and IL-17A production

Studies by our group and others demonstrating that Th17 cells are highly permissive to HIV-1 infection35,36,37 prompted us to investigate the effect of metformin on Th17 polarization. Results show that metformin acted on memory CD4+ T cells from uninfected participants to increase the frequency and intensity of RORC2 and CCR6 expression at single-cell level, enhanced the frequency of RORC2+CCR6+ T cells (Figures S2A–S2F), and maintained T cell capacity to produce IL-17A in response to TCR triggering upon culture in vitro (Figures S2G and S2H). In contrast, INK128 did not increase RORC2 expression and the frequency of RORC2+CCR6+ T cells (Figures S2A–S2F), and decreased IL-17A production early upon TCR triggering (Figures S2G and S2H). Thus, the effects of metformin on cell-associated HIV-p24 expression coincide with the promotion of a Th17 phenotype and the preservation of Th17 effector functions in memory CD4+ T cells.

Effects of metformin on CD4, CCR5, and CXCR4 expression

To get insights into the molecular mechanisms underlying differences between metformin and INK128, cells were analyzed for the expression of the HIV-1 entry receptor CD4, and co-receptors CCR5 and CXCR4. Metformin did not impact on CD4 and CXCR4 surface protein expression, while INK128 slightly decreased CD4 and increased CXCR4, mainly in terms of geometric MFI at single-cell level (Figures S3A and S3B). Levels of CCR5 mRNA expression tended to decrease, with differences reaching statistical significance in matched comparisons for INK128, but not metformin versus medium (Figure S3C). Thus, the effects of metformin on HIV-1 replication appear independent of the modulation of viral entry receptors and co-receptors.

Metformin facilitates HIV-1 replication post-integration and prior to viral release

To localize the step(s) of the HIV-1 replication cycle affected by metformin, single-round infection with a VSVG-pseudotyped HIV-1 (HIVVSV-G), with GFP in place of Env, but encoding for functional Nef and Vpu, was performed on memory CD4+ T cells from HIV-uninfected participants in the presence/absence of metformin (1 mM) or INK128 (50 nM) (Figure 2A). In agreement with results in Figure 1, metformin did not significantly modify levels of early and late reverse transcripts, nor HIV-DNA integration (Figures 2B–2D), supporting the idea that metformin acts at post-integration level(s). In contrast, INK128 significantly reduced levels of RU5 and Gag HIV-DNA (Figures 2B and 2C) metformin slightly increased the frequency of CD4lowHIV-p24+ T cells without significantly enhancing the intensity of HIV-p24 expression per cell (Figures 2E–2G). In this model of single-round infection, where cell-to-cell transmission does not occur given the absence of Env on the surface of progeny virions, the CD4highHIV-p24+ T cell population was not observed (Figure 2E), likely since Env-deficient virions cannot bind to new cells. Similar to results in Figure 1, metformin did not proportionally increase the HIV-p24 release in cell-culture supernatants (Figure 2H). In contrast, INK128 diminished the intensity of HIV-p24 expression at single-cell level, although it exerted no effect on HIV-p24 levels in cell-culture supernatants (Figures 2F–2H). These results suggest that metformin acts at post-integration levels by facilitating cell-associated HIV-p24 expression, with no effect on viral release.Figure 2 Effects on metformin on single-round HIV-1 infection in vitro

(A) Shown is the flow chart for the single-round HIV-1 infection in vitro. Briefly, memory CD4+ T cells from HIV-uninfected donors were stimulated by anti-CD3/CD28 Abs in the absence/presence of metformin (1 mM) or INK128 (50 nM) for 3 days. Then, cells were exposed to a replication-incompetent single-round VSV-G-pseudotyped HIV-1 construct (100 ng HIV-p24/106 cells). Cell-culture supernatants and cells were collected at day 3 post-infection. Shown are levels of early (RU5) (B) and late HIV reverse transcripts (Gag) (C), as well as integrated HIV-DNA (D) quantified by real-time nested PCR. Shown are representative flow cytometry dot plots of intracellular HIV-p24 and surface CD4 expression from one donor (E) and statistical analysis of the productively infected CD4lowHIV-p24+ T cells in terms of frequencies (F) and the geometric MFI of HIV-p24 expression (G). Shown are absolute HIV-p24 levels in cell culture supernatants quantified by ELISA (H). Each symbol represents one donor (n = 7; median ± interquartile range). Friedman and uncorrected Dunn’s multiple comparison p values are indicated on the graphs.

Metformin promotes VR reactivation in memory CD4+ T cells of ART-treated PWH

To explore the effect of metformin (1 mM) on the reactivation of HIV-1 reservoirs that persist during ART in PWH, we performed a simplified viral outgrowth assay (VOA),38 as depicted in Figure 3A, using cells from ART-treated PWH (n = 11; Table 1) (Figure 3). In the absence of ARVs, the mTOR inhibitor INK128 significantly reduced viral outgrowth, as reflected by levels of integrated HIV-DNA in cells and HIV-p24 levels in cell-culture supernatants, as well as intracellular HIV-p24 expression (Figures 3B–3E, left panels), consistent with the antiviral effects of INK128 previously reported by our group and others.15,20 Unexpectedly, under these same experimental conditions, metformin did not decrease HIV-DNA integration at day 12 in culture, nor viral release in cell-culture supernatants (Figures 3B and 3C, left panels). In contrast, a tendency for a metformin-mediated increase in HIV-DNA integration was observed, with median levels changing from 4,478 to 11,830 integrated HIV-DNA copies/106 T cells in the absence and the presence of metformin, respectively (Figure 3B, left panels). Similarly, HIV-p24 levels in cell culture supernatants were slightly increased by metformin (p = 0.0788) (Figure 3C, left panels). Metformin robustly increased the frequency of productively infected cells, identified as cells with a CD4lowHIV-p24+ phenotype (Figures 3D and 3E, left panels). Metformin also increased the frequency of a relatively small subset of CD4highHIV-p24+ T cells (Figures 3D and 3F, left panels). There was no increase in the geometric MFI of HIV-p24 expression within CD4lowHIV-p24+ and CD4highHIV-p24+ T cells (Figure 3G, left panel). These results demonstrate that metformin facilitates the expansion of productively infected cells upon TCR-triggering in vitro, without a proportional increase in the release of free progeny virions in cell-culture supernatants as observe in the context of HIV infection in vitro.Figure 3 Effects of metformin on viral outgrowth in memory CD4+ T cells from ART-treated PWH

(A) Shown is the flow chart for the viral outgrowth assay (VOA). Briefly, memory CD4+ T cells from ART-treated PWH were stimulated with CD3/CD28 Abs, in the presence (n = 11) or the absence (n = 6) of ARVs and in the presence/absence of metformin (1 mM) or INK128 (50 nM) for 3 days. Supernatants were collected, cells were split in two news wells, and media containing IL-2 and metformin or INK128 was refreshed every 3 days. Experiments were performed in 4–8 original replicates per condition. One original replicate at day 0 generated 8 final replicates at day 12. At the end of the experiment, T cells derived from the same original replicate were merged for PCR and flow cytometry analysis. Shown are (B) integrated HIV-DNA levels quantified by real-time nested PCR and (C) levels of HIV-p24 in cell-culture supernatants quantified by ELISA. Shown are (D) representative flow cytometry dot plot of cell-associatedHIV-p24 and surface CD4 expression; (E) statistical analysis of the frequency of CD4lowHIV-p24+, (F) the frequency of CD4highHIV-p24+ cells; and (G) the geometric MFI (GeoMFI) of HIV-p24 expression in CD4low HIV-p24+ and CD4high HIV-p24+ T cell subsets. Each symbol represents one donor; bars indicate the median ± interquartile range. Kruskal-Wallis test and uncorrected Dunn’s multiple comparison p values are indicated on the graphs.

Table 1 Clinical parameters of ART-treated PWH participants

ID	Sex	Ethnicity	Age&	CD4#	CD8 #	Plasma VLa	Time since infectionb	ART regimen	Time on ARTb	
ART #1	M	Caucasian	36	542	803	<40	13	Stribild	12	
ART #2	M	Caucasian	49	458	899	<40	201	Truvada
Viramune	201	
ART #3	M	Caucasian	58	546	1,116	<40	408	Atripla	369	
ART #4	M	Caucasian	44	546	775	<40	154	Complera	25	
ART #5	M	Caucasian	51	546	1,322	<40	149	Sustiva
Truvada	149	
ART #6	M	Caucasian	33	546	854	<40	89	Stribild	77	
ART #7	M	Caucasian	21	546	399	<40	8	Stribild	4	
ART #8	M	Caucasian	47	546	1,156	<40	182	Atripla	63	
ART #9	M	Caucasian	59	546	836	<40	273	Symtuva	273	
ART #10	M	Caucasian	64	546	620	<40	186	Triumeq	186	
ART #11	M	Latino-American	31	546	1,000	<40	69	Complera	N/A	
ART #12	M	Caucasian	30	546	605	<40	80	Stribild	77	
ART #13	F	Caucasian	31	546	445	<40	212	Viracept
Truvada	187	
ID, participant identification code; ART, antiretroviral treated PWH; M, male; F, male; &, years; #, counts of cells/μL blood; N/A, not available.

a HIV-RNA copies per mL of plasma.

b Months.

For a fraction of 6 out of 11 ART-treated PWH, experiments were performed in parallel in the presence of ARVs (integrase inhibitor Raltegravir; protease inhibitor Saquinavir), to block the infection of new cells by progeny virions produced upon TCR-mediated VR reactivation (Figure 3). Results in Figure 3B reveal, a strong decrease in integrated HIV-DNA levels mediated by ARVs in all three conditions, with the abrogation of differences between medium and metformin versus INK128 observed in the absence of ARVs. Similarly, in the presence of ARVs, soluble and cell-associated HIV-p24 levels were dramatically decreased in all three conditions (Figures 3C–3E). Thus, the proviral effects of metformin were abrogated in the presence of ARVs, which block cell-to-cell transmission of virions newly produced by reactivated VR in vitro. Together these results support a model in which metformin exerts its proviral effects by facilitating cell-to-cell transmission independently of cell-free virion release, consistent with previous reports.39,40

To get more mechanistic insights into the metformin mechanism of action in the VOA performed in the absence of ARVs, cells harvested at day 12 post-TCR triggering were analyzed for the expression of RORC2, CCR6, and IL-17A. Metformin versus medium increased the frequency of RORC2+ and CCR6+ T cells and the intensity of RORC2 and CCR6 expression at single-cell level (Figures S4A–S4C). Also, metformin versus medium slightly increased the frequency of CD4+ T cells co-expressing RORC2 and CCR6, identified as Th17-like cells (Figures S4A and S4D), but had no effect IL-17A production in cell-culture supernatants (Figure S4E). For INK128 versus medium, despite an increase in the frequency of cells expressing CCR6 (Figures S4A and S4C), and a tendency for an increase in the frequency of RORC2-expressing cells (p = 0.0856) (Figures S4A and S4B), there was a significant reduction in IL-17A production in cell-culture supernatants (Figure S4E). Thus, metformin increased the frequency of CD4+ T cells with a Th17 phenotype (Figure S4D), without proportionally increasing their effector functions (i.e., IL-17A production).

These results reveal that, in contrast to INK128 that inhibits both HIV-1 outgrowth and IL-17A production, metformin increases the frequency of T cells expressing cell-associated HIV-p24 via mechanisms independent on free-virion release and maintains Th17 functions. These observations raise new questions on the effects of metformin on specific post-integration steps of the HIV-1 replication cycle.

Metformin increases surface expression of BST2 on productively infected T cells in vitro

HIV-1 release is controlled by complex mechanisms, including BST2, a protein that sequester newly formed viral particles at the cell-surface membrane.41,42 The HIV-1 accessory protein Vpu counteracts the effects of BST2 by inducing its downregulation42,43 or displacement from the site of viral assembly,44 with BST2 mediating cell-to-cell transmission of Vpu-defective HIV-1 virions.39 Considering the discrepancy between the effects of metformin on the frequency of productively infected cells and virion release, we hypothesized that metformin limits virion release and facilitate their cell-to-cell transmission by promoting BST2 expression. To test the possibility, memory CD4+ T cells harvested at day 12 post-infection with HIV-1 NL4.3Bal in vitro (Figure 1A) and memory CD4+ T cells of ART-treated PWH harvested at day 12 post-TCR triggering (Figure 3A) were analyzed for surface expression of BST2 and cell-associated HIV-p24. Results in representative donors depicted in Figure 4A reveal the typical down regulation of BST2 on productively infected T cells. Further, the expression of BST2 was analyzed on productively infected (CD4lowHIV-p24+) versus uninfected (CD4+HIV-p24-) T cells upon HIVNL4.3BaL infection in vitro (Figures 4B and 4C), in VOA (Figures 4D and 4E), and in uninfected CD4+ T cells (Figure 4F). Upon HIVNL4.3BaL infection in vitro, metformin and INK128 significantly increased BST2 expression at the surface of productively infected, but not uninfected T cells (Figures 4B and 4C). In contrast, INK128 but not metformin increased surface BST2 expression on productively infected T cells in VOA (Figures 4D and 4E). The upregulation of BST2 was not observed when uninfected memory CD4+ T cells were exposed to metformin or INK128 (Figure 4F).Figure 4 Metformin increases BST2 expression on productively infected CD4lowHIV-p24+ T cells

(A) Shown are representative flow cytometry dot plots of BST2 and HIV-p24 co-expression on T cells at day12 post-infection with HIV-1NL4.3 Balin vitro (upper panel) and at day 12 post TCR-mediated VR reactivation in VOA (bottom panel).

(B and C) The HIV-1NL4.3Bal infection in vitro was performed as described in Figure 3. Cells collected at day 12 post-infection were stained on the surface with CD4 and BST2 antibodies and with HIV-p24 antibodies and analyzed by flow cytometry for n = 8. Shown are (B) levels of BST2 expression on uninfected (CD4highHIV-p24-) and productively infected (CD4lowHIV-p24+) T cells in one representative donor and (C) statistical analysis of BST2 expression (GeoMFI) relative to the medium condition (considered 1).

(D and E) The VOA was performed as described in Figure 1 with cells from n = 6 ART-treated PWH. Cells collected at day 12 post-stimulation were stained on the surface with CD4 and BST2 Abs and intracellularly with HIV-p24 Abs and analyzed by flow cytometry.

(D and E) Shown are levels of BST2 expression on CD4highHIV-p24- and CD4lowHIV-p24+ T cells in (D) one representative donor and (E) statistical analysis of BST2 expression (absolute GeoMFI).

(F) Shown is the BST2 expression relative to the medium condition of HIV-uninfected memory CD4+ T cells at day 3 post-TCR stimulation. Each symbol represents one donor (median ± interquartile range). Friedman (C and F), Kuskal-Wallis (E) and uncorrected Dunn’s multiple comparison p values are indicated on the graphs.

These results reveal that metformin prevents the HIV-mediated downregulation of surface BST2 expression on productively infected cells, only upon exposure to HIV-1 in vitro, while INK128 demonstrated to be a robust modulator of surface BST2 expression in VOA as well. The fact that metformin and INK128 failed to modulate BST2 expression in the absence of HIV-1 exposure points to an HIV-dependent mechanisms of action for metformin.

Metformin increases intracellular Bcl-2 expression

We previously reported that 12 weeks of metformin treatment in complement of ART in PWH increased Bcl-2, an anti-apoptotic and pro-survival marker, in colon CCR6+ CD4+ T cells.27 We therefore investigated whether metformin increases the frequency of productively HIV-infected T cells by promoting their survival in a Bcl-2-dependent manner. Memory CD4+ T cells harvested at day 12 post-infection with HIV-1 NL4.3Bal in vitro were analyzed for intracellular Bcl-2 expression by flow cytometry on productively infected (CD4lowHIV-p24+) versus uninfected (CD4highHIV-p24-) T cells (Figure 5A). Metformin, but not INK128, significantly increased the expression of Bcl-2 in both productively HIV-infected and uninfected CD4+ T-cells (Figure 5B). These results suggest that metformin treatment promotes cell survival, in line with the use of metformin as a senolytic drug.45Figure 5 Metformin increases Bcl-2 expression on HIV-infected and uninfected T cells

The HIV-1NL4.3Bal infection in vitro was performed as described in Figure 3. Cells collected at day 12 post-infection were stained with CD4, HIV-p24, and Bcl-2antibodies and analyzed by flow cytometry. Shown are (A) levels of Bcl-2 expression on uninfected (CD4highHIV-p24-) and productively infected (CD4lowHIV-p24+) T cells in one representative donor and (B) statistical analysis of Bcl-2 expression (GeoMFI) relative to the medium condition (considered 1). Each symbol represents one donor (n = 4; median ± interquartile range). Friedman and uncorrected Dunn’s multiple comparison p values are indicated on the graphs.

Metformin facilitates recognition of reactivated VR by HIV-Env antibodies

To determine whether metformin may be used to accelerate VR decay in ART-treated PWH, we hypothesized that metformin increases the recognition of reactivated VR by HIV-1 envelope (Env) Abs, thus facilitating their purging via Abs-dependent mechanisms. To test this hypothesis, we performed a VOA, as described in Figure 3A. Cells harvested at day 12 post-TCR triggering in the presence or the absence of metformin were stained on the surface with CD4 Abs and a set of broadly neutralizing (bNAbs; 2G12, PGT121, PGT126, PGT151, 3BNC117, 101074, VRC03) and non-neutralizing (nnAbs; F240, 17b, A32) HIV-1 Env Abs, and with cell-associated HIV-p24 KC57 Abs (Figure 6A). The analysis was performed using a gating strategy depicted in Figure S5A. Different HIV-1 Env Abs showed a distinct ability to bind on productively infected cells (CD4lowHIV-p24+) exposed or not to metformin (Figure 6A). Statistical analysis revealed that metformin increased the recognition of CD4lowHIV-p24+ by the PGT126 bnAbs, in terms of frequency and mean fluorescence intensity (Figures 6B and 6C). PTG126 bnAbs recognizes the “closed” conformation of the HIV-1 Env (high-mannose patch on HIV gp120),46 indicative that, upon TCR-mediated reactivation of VRs in ART-treated PWH, metformin increased the surface expression of HIV-1 Env in its “closed” conformation. Finally, considering the principle of HIV-Flow assay, where two distinct HIV-p24 Abs were used to specifically identify productively infected T cells,47 further the frequency of CD4low T cells co-expressing HIV-p24 and HIV-Env on CD4low cells was analyzed within live cells (Figure S5B). Staining performed in parallel on cells from one HIV-uninfected donor showed low/undetectable levels of non-specific HIV-p24 and HIV-Env Ab binding (Figure S5C). Metformin significantly increased the frequency of T cells expressing HIV-p24 co-recognized by multiple HIV-Env Abs (i.e., PGT121, PGT126, PGT151, 101074, F240, A32) (Figure 6D). The dual staining with HIV-p24 and HIV-Env Abs likely represents a new strategy for the identification of VRs detectable by the immune system. Whether this recognition could translate in potential killing of infected cells by Ab-dependent cell toxicity (ADCC) remains to be determined.Figure 6 Metformin facilitates the recognition of reactivated HIV reservoirs by anti-HIV Env antibodies

The VOA was performed on memory CD4+ T cells from ART-treated PWH, as described in Figure 3A. Cells harvested at day 12 post-TCR triggering and cultured in the presence/absence of metformin (1 mM) were stained on the surface with a set of unconjugated human anti-HIV-1 Env bNAbs (2G12, PGT121, PGT126, PGT151, 3BNC117, 101074, VRC03) and nnAbs (F240, 17b, A32), followed by incubation with anti-human Alexa Fluor 647-conjugated secondary Abs. Further, cells were stained on the surface with CD4 Abs, as well as intracellularly with HIV-p24 Abs. (A) Show are flow cytometry dot plot representations of HIV-Env Ab binding on productively infected CD4lowHIV-p24+ T-cells in one representative donor, gated as depicted in Figure S5A. Statistical analysis were performed to determine the effect of metformin on the frequency of CD4lowHIV-p24+ T cells recognized by anti-HIV-Env Abs (B), as well as the geometric MFI of HIV-Env expression (C). Finally, statistical analysis were performed to determine the effect of metformin on the frequency of T cells co-expressing cell-associated HIV-p24 and surface HIV-Env (D), gated as in Figures S5A and S5B. Wilcoxon test p values are indicated on the graphs. Each symbol represents one donor (n = 8; median ± interquartile range).

Discussion

Although ART has saved and substantially improved the life of PWH, the treatment is not curative and chronic HIV-1 infection is associated with several comorbidities that represent a global health burden.1,8 New therapeutic strategies are needed to reduce chronic inflammation, improve immune functions, and accelerated the decay of VRs. HIV-1 infection modifies the metabolism of immune cells and thus, cellular metabolism could be a potential target for HIV-1 cure interventions.48 By using metformin, an FDA approved anti-diabetic drug that reduces mTOR pathway activity,25 we expected to reduce HIV-1 replication. Unexpectedly, metformin treatment increased the frequency of productively infected T cells in the context of a VOA performed with memory CD4+ T cells from ART-treated PWH, as well as upon HIV-1 infection in vitro. Nevertheless, the increase in the frequency of productively HIV-infected T cells did not lead to a proportional increase in progeny free-virion release. These effects were associated with increased expression of BST2 and/or Bcl-2 on productively infected T cells. Finally, the finding that metformin promotes the immune recognition by HIV-Env Abs (i.e., bnAbs PGT126) of T cells carrying translation-competent VRs emphasize the potential beneficial effect of metformin in accelerating the decay of VRs in ART-treated PWH in the context of efficient HIV-Env Abs-mediated antiviral responses, such as ADCC.49

In this study, we used three viral models that allowed us to dissect specific steps of the viral replication cycle affected by metformin. In the VOA, the outgrowth of replication-competent virions was quantified in memory CD4+ T cells of ART-treated PWH upon culture in vitro, with the genetic features of VR proviruses (mutations, deletions) remaining unknown. For in vitro infections, we used the HIVNL4.3BaL molecular clone, a wild-type replication-competent CCR5-tropic HIV-1, and the single-round HIVVSV-G construct contained an EGFP gene inserted in the Env gene, leading to the generation of Env-defective HIV-1 virions. In all three models, metformin treatment did not increase viral release but increased the frequency of productively infected CD4lowHIV-p24+ T cells. Mechanisms by which metformin increased the frequency of productively HIV-infected cells, without a proportional viral release in all three viral model remains to be evaluated. In the replication competent HIVNL4.3BaL infection in vitro, metformin increased cell-associated HIV-p24, decreased free-virion release, and prevented the HIV-induced downregulation of BST2, a phenomenon that we did not observe in the two other experimental models. Metformin effects were less pronounced in experiments performed with HIVVSV-G, in line with the inability of Env-defective viruses to be tethered at the cell-surface via BST2 and to infect new cells. Infection using a single round VSVG pseudotyped HIV virus showed that metformin acts on the post-integration steps of the HIV replication cycle, such as translation, assembly, budding, and/or free-virion release. HIV transcription was not evaluated in the current studies. Metformin effects in VOA were abrogated by ARVs, further supporting the idea of a metformin-mediated mechanism of cell-to-cell transmission independent of free-virion release. These findings suggest that metformin facilitates the reactivation of VR cells.

mTOR controls Th17 polarization and functions.50,51 Direct mTOR inhibitors such as INK128 and rapamycin reduce IL-17A production.52,53 Metformin was previously reported to reduce IL-17A production and RORC2 expression in vitro.54 The latter report contrasts with results from our study, likely because the latter study was performed under Th17-polarizing conditions. Our results show that metformin treatment, in contrast to INK128, promotes CCR6 and RORC2 protein expression and maintains the Th17 cell effector functions (i.e., IL-17A). Th17 cells are largely depleted after HIV-1 infection and their maintenance is linked to a better control of HIV-1 replication in elite controller.53,55 Furthermore, studies by our group demonstrated that Th17 cells are highly susceptible to HIV-1 infection given their unique high metabolic activity and transcriptional profiles.20,35,36,56,57 The pleiotropic effects of metformin on various steps of the viral replication cycle coincided with an increased Th17-polarization phenotype (CCR6+RORC2+), and a preserved IL-17A production. Therefore, metformin treatment could exert its proviral activities by boosting Th17 polarization.

Another major finding of our study is the demonstration that metformin increased the expression of BST2, a host-cell restriction factor originally reported to tether progeny virions on the cell surface, thus preventing their release.42 Further studies reported that basal levels of BST2-mediated virion tethering are required for efficient cell-to-cell transmission of HIV-1,40 mainly in primary cells.58 Indeed, we found that, upon HIV-1 exposure in vitro, metformin reduced cell-free virion levels, while enhancing the frequency of productively infected cells and boosting their BST2 expression. In contrast to metformin, INK128 decreased cell-surface BST2 expression. Considering that BST2 is a viral restriction factor induced by type I interferon41 and that mTOR activation induces interferon production,59 we hypothesized that INK128-mediated mTOR blockade reduced BST2 expression in an interferon-dependent manner. The antiviral features of BST2 are regulated via glycosylation, intracellular trafficking and epigenetic modifications.60,61,62 BST2 exists in two isoforms, long (L-tetherin) and short (S-tetherin), with Vpu mainly targeting the long isoforms.63 The fact that BST2 acts through an interaction with the HIV-1 Env,42,64 explains the accumulation of cell-associated HIV-p24 in T cells upon exposure to wild type HIVNL4.3 Bal but not Env-deficient VSVG-pseudotyped HIV-1. The HIV-1 accessory protein Vpu facilitates viral release by decreasing BST2 expression and its restriction activity,42 pointing to the possibility that metformin counteracts the Vpu-mediated BST2 downregulation on infected T cells. The ability of Vpu to counteract BST2 depends on its serine phosphorylation,65 a process that may be modulated by metformin in a mTOR-dependent manner. Additionally, activation of the PI3K signaling pathway, which acts upstream to activate the mTOR pathway, results in reduced cell-surface expression of BST2 in macrophages,66 thereby suggesting a potential role of metformin in regulating BST2 expression via a mTOR-dependent mechanism. In VOA, BST2 expression on productively infected cells was not influenced by metformin, consistent with the fact that a metformin-mediated increase in cell-associated HIV-p24 expression was not observed at single cell level in the VOA. This raises new questions on the specificity of metformin action in relationship with the particularities of reactivated proviruses. Molecular mechanisms by which metformin regulate BST2 expression and functions (e.g., transcription of specific isoforms, glycosylation, cellular localization, modulation of Vpu expression, epigenetic modifications) remain to be further elucidated.

The effects of metformin on the expansion of productively infected T cells upon infection in vitro were also associated with an increased expression of Bcl-2, a mitochondrial protein associated with cell survival.67 The upregulation of Bcl-2 by metformin on colon-infiltrating CCR6+CD4+ T cells was also observed in our LILAC pilot clinical trial.27 Most recent studies demonstrated the clinical benefits of Bcl-2 inhibitors (i.e., Venetoclax) in promoting VR purging.68,69,70,71 Whether metformin supplementation of ART may render reactivated VR more sensitive to Bcl-2 blockade, requires investigations in in vivo models.

In HIV eradication strategies, both the “shock” and “kill” arms will be required.72,73 BST2 acts as an innate sensor of viral assembly,74 suggesting that metformin may facilitate VR sensing by the immune system via BST2-dependent mechanisms. Indeed, studies by our group and others documented the ability of BST2 to facilitate ADCC.75,76,77,78,79,80 In this context, we assessed the impact of metformin on the recognition of reactivated VRs by bNAbs and nnAbs. We demonstrated that metformin treatment increased the frequency of productively infected CD4+ T cells recognized by the bNAbs PGT126, as well as its binding intensity at the single-cell level. The antiviral activities of PGT126 bNAbs were tested in a rhesus macaque infection model, in which PGT126 Abs administered before vaginal or rectal SHIV challenge displayed protective effects against infection acquisition.76,81 Whether metformin can increase the ADCC activity of PGT126 Abs for infected cells remains to be demonstrated. If so, a combination therapy including metformin and bNAbs could be beneficial to reduce the size of HIV-1 reservoirs during ART. In addition to ADCC mediated by NK cells, CD8+ T cells are also key effectors for the control HIV-1 replication.82,83 Of interest, CD8+ T cells differentiation and antiviral functions are dependent on the mTOR activity.84 In this context, the role of metformin treatment on CD8+ T cell-mediated killing of HIV-infected T cells remain to be elucidated. In line with this possibility, studies in tumor cell lines demonstrated that metformin increased the cytotoxic activities of CD8+ T cells against cancerous cells.85 Of note, eight weeks of metformin treatment in nondiabetic PWH increased the cytotoxic response of CD8+ T cells.86 These data support the idea that metformin could have a beneficial effect both on HIV-1 reservoir reactivation and on the quality of HIV-specific CD8+ T cell responses.

In conclusion, our results support a model in which metformin supplementation of ART facilitates the expression of HIV-p24, helps tether the progeny virions on the cell surface via a BST2 dependent mechanism and promote their enhanced recognition by HIV-1 Env bnNAbs (i.e., PGT126). Considering its pleiotropic pro/antiviral effects on specific steps of the HIV-1 replication cycle, long-term double blind clinical trials should be performed to test metformin together with HIV-1 Env bNAbs in complement of ART in PWH as a novel HIV-1 remission/cure strategy to target HIV reservoirs.

Limitations of the study

Although the optimal metformin concentrations used in our in vitro study (1 mM) were identified based on dose-response testing, it may not reflect the actual concentration in tissues upon metformin administration in clinic.27 Since metformin, taken orally, mostly acts in tissues such as the intestines and liver, we cannot guarantee that the effects observed in vitro on peripheral blood CD4+ T cells may be consistent with the reality in vivo. However, our decision to use metformin at 1 mM is justified by its effect on mTOR activation and HIV-1 replication, without affecting cell viability and proliferation (Figure 1; Figure S1). Also, studies by other groups also reported results using the same concentration of metformin in vitro.28,54 To decipher the molecular mechanisms used by metformin to reactivate HIV-1 reservoirs in VOA, the step of HIV transcription requires detailed investigations. Future studies on T cells enriched in VRs, such as CCR6+ Th17 cells,36,37 may be required for an accurate monitoring of metformin effect on HIV transcription. Also, chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR) studies should investigate the effect of metformin on the recruitment of transcription factors and cofactors to the HIV promoter, using approaches we recently reported.87 The flow cytometry staining of cell-associated HIV-p24 did not allow to distinguish whether virions were trapped in the cytoplasm, at the inner or at the outer cell surface membrane. Further investigation using microscopy visualization should be performed.

Finally, our studies were mainly performed on cells from Caucasian male participants with HIV-1 clade B infection (i.e., 1 female, 12 males; 1 Latin-American, 12 Caucasians). Differences linked to sex, gender, ethnicity, and HIV clade could have an impact on the effect of metformin treatment. Indeed, non-AIDS comorbidities and metabolic disorders vary depending on sex and ethnicity.88 These aspects should be further investigated in an effort to implement precision medicine strategies.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Antibodies	
	
Purified no azide (NA)/low endotoxin (LE) Mouse Anti-Human CD3 (Clone UCHT1)	BD	Cat#555329; RRID: AB_395736	
Purified NA/LE Mouse Anti-Human CD28 (Clone CD28.2)	BD	Cat#555725; RRID: AB_396068	
Mouse anti-human CD3 Pacific Blue (Clone UCHT1)	BD	Cat#558117; RRID: AB_397038	
Mouse anti-human CD4 Alexa Fluor (Clone RPA-T4)	BD	Cat#561030; RRID: AB_10563215	
Mouse anti-human CD8 FITC (Clone 130-080-601)	Miltenyi	Cat#130-080-601; RRID: AB_244336	
Mouse anti-human CCR6 PE (Clone 11A9)	BD	Cat#559562; RRID: AB_397273	
Mouse anti-human CD45RA allophytocyanin hilite (APC-H7) (Clone HI100)	BD	Cat#560674; RRID: AB_1727497	
Mouse anti-human CXCR4 APC (Clone 12G5)	eBiosciences	Cat#17-9999-42; RRID: AB_1724113	
Mouse anti-human CD317 (BST-2) BV421 (Clone Y129)	BD	Cat#566381; RRID: AB_2744363	
Hamster anti-human Bcl-2 FITC	BD	Cat#554234; RRID: AB_395319	
HIV-1 core (p24) antigen-FITC (Clone KC57)	Beckman Coulter	Cat#6604665	
HIV-1 core (p24) antigen-RD1 (Clone KC57)	Beckman Coulter	Cat#6604667; RRID: AB_1575989	
Mouse anti-human RORC2 Alex Fluor 647 (Clone Q21-559)	BD	Cat#563620; RRID: AB_2738324	
Mouse anti-human IL-17A PE (Clone eBio64DEC17)	eBiosciences	Cat#12-7179-42; RRID: AB_1724136	
Goat anti-Human IgG Secondary Antibody, Alexa Fluor 647	Invitrogen	Cat#A-21445; RRID: AB_2535862	
Anti-phospho-Ribosomal Protein S6 (Ser240/Ser244)	EMD Millipore	Cat#07 2113	
Phospho-mTOR (Ser2448)	Cell Signaling	Cat#2971; RRID: AB_330970	
Secondary antibody anti-Rabbit IgG HRP-linked	Cell Signaling	Cat#7074; RRID: AB_2099233	
Monoclonal Mouse Anti-β-Actin antibody	Millipore Sigma	Cat#A5441; RRID: AB_476744	
Goat anti-Mouse IgG (H + L) Secondary Antibody, HRP	Invitrogen	Cat#32430; RRID: AB_1185566	
	
Bacterial and virus strains	
	
One Shot Stbl3 Chemically Competent E. coli	Life Technologies	C737303	
NL4.3BaL HIV plasmid	From Dr. Michel tremblay, Université Laval, Québec, Canada	N/A	
pHEF Expressing Vesicular Stomatitis Virus (VSV-G) plasmid	NIH HIV Reagent Program, Division AIDS, NIAID (Contribution of Dr. Lung Ji Chang)	Cat#ARP-4693	
HIV-1 NL4-3 ΔEnv EGFP Reporter Vector	NIH HIV Reagent Program, Division of AIDS, NIAID (Contribution Dr. Haili Zhang, Dr. Yan Zhou and Dr. Robert Siliciano)	Cat#ARP11100	
	
Biological samples	
	
Leukaphereses of ART treated people living with HIV and HIV-uninfected people	Recruited at the Montreal Chest Institute, McGill University Health Center and Center Hospitalier de l’Université de Montréal with the help of Dr Jean-Pierre Routy’s group	N/A	
	
Chemicals, peptides, and recombinant proteins	
	
rhIL-2	R&D Systems	Cat#202-IL-050	
1,1-Dimethylbiguanide, Hydrochloride (Metformin)	Santa Cruz	Cat#sc-202000	
Sapanisertib (INK128)	Cayman Chemical	Cat#11811-1	
Saquinavir	NIH HIV Reagent Program, Division of AIDS, NIAID (Contribution DAIDS/NIAID)	Cat#ARP-4658	
Raltegravir	NIH HIV Reagent Program, Division of AIDS, NIAID (Contribution DAIDS/NIAID)	Cat#ARP-11680	
Radioimmunoprecipitation Assay Buffer (RIPA) 10X	Cell Signaling	Cat#9806S	
Phosphatase Inhibitor (PhosSTOP)	Milipore Sigma	Cat#4906845001	
Complete, Mini, EDTA-free protease inhibitor	Milipore Sigma	Cat#11836170001	
Clarity and Clarity Max ECL Western Blotting Substrates	Bio-Rad	Cat#1705062S	
ReBlot Plus Strong Antibody Stripping Solution	Sigma	Cat#2504	
Sodium Bicarbonate (NaHCO3)	Sigma	Cat#S5761	
Sodium Carbonate (Na2CO3)	Sigma	Cat#223530-500G	
Thimerosal	Sigma	Cat#T5125-10G	
Phosphate Buffered Saline (PBS)	Thermo Fisher	Cat#10010023	
Tween 20	Fisher Scientific	Cat#BP337-500	
Triton X-100	Sigma	Cat#X100-500mL	
Trypan Blue	Thermo Fisher	Cat#15250061	
Bovine Serum Albumin (BSA)	BioShop	Cat#ALB001.500	
Streptavidin Horseradish Peroxidase (Strep-HRP)	Fisher Scientific	Cat#65R-S104PHRP	
3,3′,5,5′-Tetramethylbenzidine (TMB)	Quimigen	Cat#42R-TB10265R-S104PHRP	
Phosphoric Acid (H3PO4)	Sigma	Cat#PX0996	
Nonidet P-40	Bioshop	Cat#NON505	
Tris HCl	BioShop	Cat#TRS002.500	
Proteinase K	Fisher Scientific	Cat#25530-015	
Molecular Grade Water (H2O)	Wisent	Cat#809-115-CL	
Guanidine-HC;	Promega	H5381	
Trypsin EDTA with phenol red	Wisent	Cat#075-350	
Methanol 99,98%	Fisher Scientific	Cat#BPA4084	
4X Laemmli Sample Buffer	Bio-Rad	Cat#1610747	
2-Mercaptoethanol	Sigma	Cat#M6250	
Precision Plus Protein Dual Color Standards	Bio-Rad	Cat#1610374	
Glycine	BioShop	Cat#GLN001.500	
Sodium Chloride	BioShop	Cat#SOD002	
30% Bis-acrylamide Solution	Bioshop	Cat#ACR010.500	
UltraPure Tris Buffer	Thermo Fisher	Cat#15504-020	
Sodium Dodecyl Sulfate (SDS)	Bio-Rad	Cat#1610302	
N,N,N′,N′-Tetramethyl Ethylenediamine (TEMED)	Sigma	Cat#8087420005	
Ammonium Persulfate (APS)	Bioshop	Cat#AMP001.100	
Formaldehyde solution 37 wt. % in H2O	Sigma	Cat#F1635-500ML	
Sodium Azide	Bioshop	Cat#SAZ001.250	
Fetal Bovine Serum (FBS)	Wisent	Cat#091-150	
Dimethyl Sulfoxide (DMSO)	Sigma	Cat#34869-500mL	
RPMI 1640 Medium (RPMI)	Thermo Fisher	Cat#11875119	
Penicillin/streptomycin (PenStep)	Thermo Fisher	Cat#15140122	
Lymphocyte Separation Medium (LSM)	Wisent	Cat#305-010-CL	
EDTA	Bioshop	Cat#EDT001.1	
Opti-MEM	Gibco	Cat#31985070	
	
Critical commercial assays	
	
p24 Enzyme-linked Immunosorbent Assay (ELISA)	Homemade. Hybridome provided by Dr. Michel J. Tremblay (Bounou et al.89)	N/A	
QuantiTect SYBR Green RT-PCR Kit	Qiagen	Cat#204243	
10X PCR Buffer/Magnesium Chloride (MgCl2) Buffer/Thermus Aquaticus (TAQ) Polymerase	Thermo Fisher	Cat#18038067	
LC480 probe master mix	Roche	Cat#4707494001	
QuantiTect SYBR Green RT-PCR Kit	Qiagen	Cat#204243	
Deoxynucleoside Triphosphates (dNTP)	Thermo Fisher	Cat#10297018	
Wizard Plus DNA Purification System	Fisher Scientific	PR-A7100	
EndoFree Plasmid Maxi Kit	Qiagen	12362	
EasySep Memory CD4+ T cell Isolation Kit, human	StemCell Technology	Cat#19157	
Rneasy Plus Mini Kit	Qiagen	Cat#74136	
Detergent Compatible (DC) Protein Assay	Bio-Rad	Cat#5000111	
Foxp3 transcription factor staining buffer set	eBioscience	Cat # 00-5523-00	
X-tremeGENE 9 kit	Roche	Cat#06 365 779 001	
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (405 nm excitation)	Thermo Fisher	Cat#L34957	
	
Experimental models: cell lines	
	
293T	ATCC	Cat#CRL-3216	
ACH-2	NIH HIV Reagent Program	Cat#ARP-349	
	
Oligonucleotides	
	
See Table S2: Oligonucleotides	See Table S2: Oligonucleotides	See Table S2: Oligonucleotides	
	
Software and algorithms	
	
FlowJo version 10	BD	https://www.flowjo.com/	
GraphPad Prism 9.0.1	GraphPad	https://www.graphpad.com/	
Image Lab	Bio-Rad	https://www.bio-rad.com/en-ca/product/image-lab-software?ID=KRE6P5E8Z	
	
Other	
	
Immobilon-PSQ Polyvinylidene Difluoride (PVDF)	Sigma	Cat#ISEQ00010	
	
Resource availability: data availability	
	
Uncropped western blot (Figures S1A and S1C)	Mendeley Data	Fert, Augustine (2024), “Metformin Facilitates Viral Reservoir Reactivation and their Recognition by Anti-HIV-1 Envelope Antibodies. Fert et al.”, Mendeley Data, V1, https://doi.org/10.17632/trckskyvcw.1	

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Petronela Ancuta (petronela.ancuta@umontreal.ca).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• Uncropped western blot membrane displayed in Figures S1A and S1C have been deposited at the database Mendeley Data (https://data.mendeley.com) and are publicly available as of the date of publication. Accession number is listed in the key resources table.

• This study did not generate new codes

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Ethics statement

Study participants were recruited at the Montreal Chest Institute, McGill University Health Center, and Center Hospitalier de l’Université de Montréal (Montreal, Québec, Canada), in compliance with the principles included in the Declaration of Helsinki. This study received approval from the Institutional Review Board (IRB) of the McGill University Health Center and the CHUM Research Center, Montreal, Quebec, Canada. All participants signed a written informed consent and agreed with the publication of the results.

Study participants

This study was performed using Peripheral Blood Mononuclear Cells (PBMCs) from ART-treated PWH (n = 13) and HIV-uninfected (n = 15) study participants. PBMCs were isolated by gradient density centrifugation from leukapheresis and maintained frozen in liquid nitrogen until use.90 Clinical parameters of study participants are included in Table 1 for PWH and Table S1 for HIV-uninfected donors.

Memory CD4+ T cell sorting

Memory CD4+ T-cells were isolated from PBMCs of HIV-uninfected and ART-treated PWH by negative selection using the EasySep Human Memory CD4+ T cell Enrichment Kit (StemCell Technology), following the manufacturer recommendation. The cell purity after sorting was >95%, as determined upon staining with CD3, CD4, CD45RA and CD8 Abs and flow cytometry analysis (BD LSRII).

Flow cytometry analysis

For surface staining, cells were incubated for 30 min at 4°C in PBS 1X buffer containing 10% FBS (Wisent), 0.02% NaN3 and fluorescence-conjugated antibodies against CD3, CD4, CD8, CCR6, CD45RA, CXCR4 and BST2 (Table S1), using a protocol we previously reported.20,57 Live/Dead Fixable Aqua Dead cells stain Kit was used to exclude dead cells (Invitrogen). Intracellular/nuclear staining was performed using the FoxP3 transcription factor staining buffer kit (eBioscience) and fluorescence-conjugated antibodies against HIV-p24 KC57, RORC2 and Bcl-2 (key resources table). Flow cytometry acquisition of stained cells was performed on a BD LSRII cytometer. Flow cytometry analysis was performed using the BD Diva and FlowJo version 10. The positivity gate for RORC2 were placed using the fluorescence minus one (FMO) strategy. The positivity gate for HIV-p24 (KC57) were placed using uninfected memory CD4+ T-cells.

Cell culture and activation

For TCR triggering of primary memory CD4+ T-cells, cells were cultured in RPMI1640 (GIBCO) cell-culture media (10% FBS, 1% Penicillin/Streptomycin (GIBCO) at 1 × 106 cells/mL in the presence of immobilized CD3 antibodies (1 μg/mL; BD Biosciences) and soluble CD28 antibodies (1 μg/mL; BD Biosciences).

Compounds

The following drugs were used to treat primary CD4+ T-cells: metformin (0.1; 0.5; 1 and 5 mM) (1,1-Dimethylbiguanide, HydrochlorideSanta Cruz); INK128 (50 μM) (Cayman); Saquinavir (5 μM) (NIH HIV Reagent Program), and Raltegravir (0.2 μM) (NIH HIV Reagent Program).

HIV viral stocks

In this study, the following HIV-1 viruses were used (i) replication-competent CCR5 using (R5) NL4.3BAL and (ii) single-round VSVG-HIV-GFP, an env-deficient NL4.3 provirus pseudotyped with the VSV-G envelope and encoding for gfp in place of env. The NL4.3BaL HIV plasmid was provided by Michel Tremblay, Université Laval, Quebec, Canada, originating from Roger J. Pomerantz, Thomas Jefferson University, Philadelphia, PA. The plasmid pHEF Expressing Vesicular Stomatitis Virus (VSV-G) (ARP-4693) was obtained through the NIH HIV Reagent Program, Division of AIDS, NIAID, NIH, contributed by Dr. Lung-Ji Chang. The HIV vector containing the NL4-3 backbone encoding for enhanced green fluorescent protein (EGFP) in place of the Envelope (Env) (NL4.3EGFPΔEnv), and encoding for functional Nef and Vpu, was obtained through the NIH HIV Reagent Program, Division of AIDS, NIAID, NIH, contributed by Dr. Haili Zhang, Dr. Yan Zhou and Dr. Robert Siliciano. The plasmids were amplified upon bacterial transformation by MiniPrep (Promega) and MaxiPrep (Qiagen) following the manufacturer recommendation. The plasmid NL4.3Bal HIV-1 was transfected in 293T cells (ATCC) in order to produce the CCR5-tropic replication-competent NL4.3Bal HIV-1 viral stock. The 293T cell stocks were tested for Mycoplasma (ThermoFisher Scientific). The plasmids VSV-G and NL4-3 ΔEnv EGFP were transfected together in a ratio 1:3 in 293T-cells. To perform the transfection in 293T-cells, using the X-tremeGENE 9 kit (Roche), according to manufacturer’s recommendation. Cell-culture supernatant containing the virus was collected 72 h post-transfection. The NL4.3Bal HIV stock obtained on 293T-cells was passed once on TCR-activated memory CD4+ T-cells and the cell-culture supernatant was collected at day 12 post-infection. The HIV viral stocks were quantified by HIV-p24 ELISA and the quantity needed for optimal infection was determined by titration on TCR-activated memory CD4+ T-cells.

HIV-1 infection in vitro

HIV-1 infection in vitro was performed as we previously reported.57 Memory CD4+ T-cells were stimulated by CD3/CD28 Abs for 3 days prior infection. Cells were exposed to NL4.3BAL (50 ng HIV-p24/106 cells) for 3 h at 37°C and homogenized every 30 min, or VSVG-HIV-GFP (100 ng HIV-p24/106 cells) and spinoculated for 1 h at 300 g at room temperature. Unbound virions were removed by extensive washing with RPMI1640 10%FBS, 1%PS. Cells were cultured in the presence of IL-2 (5 ng/mL; R&D Systems) at 37°C for 12 and 3 days for NL4.3BAL and VSVG-HIV-GFP, respectively. A fraction of cells collected at day 3 post-infection was used for nested real-time PCR quantification of HIV-DNA. Cell-culture supernatants were harvested and productive infection was measured by HIV-p24 ELISA, using homemade Abs,89,91 and flow cytometry analysis upon surface CD4 and intracellular HIV-p24 staining. Productively infected T-cells were identified based on their CD4lowHIV-p24+ phenotype, with CD4 downregulation being indicative of productive infection.33,34

Viral Outgrowth Assay (VOA)

To measure replication-competent VRs, a simplified viral outgrowth assay was performed using a protocol developed in our lab.38 Succinctly, memory CD4+ T-cells from ART-treated PWH were cultured at 1x106cells/well in RPMI1640, 10%FBS, 1% PS cell-culture media in 48-well plates in the presence of immobilized CD3 Abs and soluble CD28 Abs (1 μg/mL). At day 3, cells were washed to remove the CD3/CD28 Abs. Cells from each well were split into two new wells for optimal cell density (typically 1-2 × 106 cells/mL/well) and cultured in the presence of IL-2 (5 ng/mL). The splitting procedure was repeated with media being refreshed every 3 days. The VOA was performed in the presence or the absence of metformin (1 mM), INK128 (50 μM) and antiretroviral drugs (ARVs; Saquinavir at 5 μM and Raltegravir at 0.2 μM). At day 12, one original replicate generated 8 splitting replicates, from which cells were harvested for the quantification of intracellular HIV-p24 expression by flow cytometry, as well as HIV-DNA by PCR. Cell-culture supernatants were collected every 3 days for HIV-p24 level quantification by ELISA.

Quantification of early, late reverse transcript and integrated HIV-DNA

Early and late HIV-1 reverse transcripts, as well as integrated HIV-DNA levels were quantified using specific primers and probes (Table S2), as we previously described.20,87 Briefly, cell lysates, generated by proteinase K digestion, were used to quantify HIV-DNA copies. Early reverse transcripts were amplified using primers specific for the RU5 region of the HIV genome, using SYBR green real-time nested PCR (Qiagen). Gag and integrated HIV-DNA, as well as CD3 DNA (used to normalize HIV-DNA expression per number of cells) were amplified using specific primers (Table S2) and nested real-time PCR. The first PCR round performed with both HIV and CD3 primers was followed by a second round of PCR performed with specific internal primers and probes on the LightCycler 480II (Roche) (Table S2). Results are expressed as HIV-DNA copies per million cells, upon normalization to CD3 copies. For all PCR quantifications, ACH2 cells (NIH HIV Reagent Program) carrying one copy of integrated HIV-DNA per cells, were used as a standard curve.

Anti-HIV-1 envelope antibodies recognition of productively HIV-infected T-cells

Memory CD4+ T-cells harvested at Day 12 of VOA were analyzed by flow cytometry for the binding of a panel human Abs directed against the HIV-1 Env. The following antibodies were used: anti-gp41 F240; anti-cluster A A32, anti-coreceptor binding site 17b; anti-CD4 binding site VRC03, 3BNC117; anti-gp120 outer domain 2G12; the gp120-gp41 interface PGT151 and anti-V3 glycan PGT121, PGT126, 101074. The goat anti-human IgGs conjugated with Alexa Fluor 647 (Invitrogen) were used as a secondary Abs to determine the levels of anti-HIV-gp120 Abs binding. Then the cells were stained on the surface with CD4-Alexa Fluor 700 Abs and intracellularly with HIV-p24-FITC Abs (key resources table). Productively infected T-cells were identified based on their CD4lowHIV-p24+ phenotype. The viability dye was used to exclude dead cells from the analysis.

HIV-1 env antibody production

FreeStyle 293F cells (Thermo Fisher Scientific) were grown in FreeStyle 293F medium (Thermo Fisher Scientific) to a density of 1 × 106 cells/mL at 37°C with 8% CO2 with regular agitation (150 rpm). Cells were transfected with plasmids expressing the light and heavy chains of A32 (kindly provided by James Robinson); VRC03 (kindly provided by John Mascola); 3BNC117, 10–1074 (kindly provided by Michel Nussenzweig); F240, 2G12, 17b (NIH AIDS Reagent Program); PGT121, PGT126, PGT151 (IAVI), using ExpiFectamine 293 transfection reagent, as directed by the manufacturer (Thermo Fisher Scientific). One week later, the cells were pelleted and discarded. The supernatants were filtered (0.22-μm-pore-size filter), and antibodies were purified by protein A affinity columns, as directed by the manufacturer (Cytiva, Marlborough, MA, USA).

Western blot

The visualization of total and phosphorylated mTOR and S6 ribosomal proteins was performed using protocols established in the laboratory.20 Cells were lysed with RIPA buffer (Cell Signaling) containing phosphatase inhibitors and protease inhibitors (Milipore Sigma) for 5 min at 4°C and sonicated 3 times for 5 s on ice. Lysed pellets were centrifuged at 14,000 g for 10 min to remove cell debris. Proteins were quantified using the kit DM Protein Assay (Bio-Rad). Loading of proteins (10 μg/well) was performed onto a 7% acrylamide SDS gel for mTOR and 15% for S6 ribosomal and electrophoretic migration was performed (1h10 at 150V). Migrated proteins were transferred by electrophoresis on activated PVDF membrane (1 h at 100V). Membranes were blocked for 45 min at room temperature with TBST 5% BSA, 0.1% Tween buffer. To measure phosphorylated proteins, membranes were bloated with primary Abs against Phosphorylated Ribosomal S6 (EMD Milipore) and Phosphorylated mTOR (Cell Signaling) Abs overnight at 4°C. Then membranes were washed with TBST 0.1 %Tween buffer and incubated with secondary antibody anti-Rabbit IgG HRP-linked (Cell Signaling) for 1 h at room temperature. Proteins were revealed with Clarity Max Western ECL Substrate, (Bio-Rad). For total mTOR and S6, the same membranes were stripped with Re-Blot Plus Strong Solution (EMD Milipore) and re-bloated with the appropriate primary and secondary Abs. For β-actin, the same membranes were stripped with Re-Blot Plus Strong Solution (EMD Milipore) and re-bloated with the primary anti-β-actin Abs (Sigma Aldrich) and HRP conjugated-secondary Abs (Invitrogen).

Statistical analysis

Statistical analyses were performed with GraphPad Prism 9.0.1. Statistical tests used are indicated in the figure legends and p-values are indicated on the graphs. P-values ≤0.05 were considered statistically significant. When the general statistical tests (i.e., Friedman) reached the statistical threshold (p-values ≤0.05), the uncorrected Dunn’s multiple comparison test was performed, with p-values indicated within the graphs to indicate statistical significance between conditions.

Supplemental information

Document S1. Figures S1–S5, Tables S1, and S2

Acknowledgments

We thank Dr. Dominique Gauchat, Philippe St Onge, and Gaël Dulude (Flow Cytometry Core Facility, CHUM-Research Center, Montréal, QC, Canada) for expert technical support; Olfa Debbeche and Laurent Knafo (Biosafety Level 3 Core Facility CHUM-Research Center). We acknowledge the key contribution of all study participants for their precious gift of leukapheresis. We thank the following collaborators for kindly providing plasmids to produce antibodies: James Robinson (Tulane University) for A32 ; John Mascola (Vaccine Research Center, NIAID) for VRC03 ; Michel Nussenzweig for 3BNC117 and 10–1074 ; the NIH AIDS Reagent Program for F240 , 2G12 , and 17b ; and the International AIDS Vaccine Initiative (IAVI) for PGT121 , PGT126 , PGT151 . The authors thank Jonathan Dias for assistance with manuscript editing.

This study was funded by grants from the Canadian Institutes of Health Research (CIHR; PJT-153052 ; PJT-178127 to P.A.), the Canadian HIV Cure Enterprise Team grant (CanCURE 1.0 ) funded by CIHR in partnership with the Canadian Foundation for AIDS Research and the International AIDS Society (CanCURE 1.0 ; HIG-133050 to P.A.), and the CanCURE 2.0 Team grant funded by CIHR (HB2-164064 to P.A.). This work was also supported by grants from the National Institutes of Health (NIH; R01 AI148379 ; R01 AI150322 ), a CIHR team grant [422148 ] and the Enterprise for Research and Advocacy to Stop and Eradicate HIV (ERASE, UM1AI164562 ) to A.Fi. and the Delaney AIDS Research Enterprise to Cure HIV (DARE, UM1AI164560 ) to A.Fi. and N.C. A.Fi. is the recipient of a Canada Research Chair on Retroviral Entry [RCHS0235 950-232424 ]. A.Fe. and D.P. received doctoral fellowship from the Université de Montréal and Fonds de Recherche Québec - Santé (FRQ-S). Core facilities and PWH cohorts were supported by the Fondation du CHUM and the FRQ-S/AIDS and Infectious Diseases Network. The funding institutions played no role in the design, collection, analysis, and interpretation of data.

Author contributions

Conceptualization, A.Fe., D.P., and P.A.; methodology, A.Fe., D.P., P.A., J.R., and A.Fi.; investigation and formal analysis, A.Fe., J.R., and L.R.M.; resources, J.-P.R., N.C., P.A., and A.Fi.; writing – original draft, A.Fe.; writing – review & editing, A.Fe., D.P., J.R., A.Fi., N.C., L.R.M., J.-P.R., and P.A.; supervision, A.Fi., N.C., and P.A.; funding acquisition, P.A.,; project administration, P.A.

Declaration of interests

The authors declare no competing interests.

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2024.110670.
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