A reservoir of latently infected cells is involved in persistence of HIV-1 during suppressive antiretroviral therapy (ART) and is responsible for viral rebound after discontinuation of treatment. Many therapeutic strategies have been proposed to reduce the size of this latent reservoir and thus achieve a functional cure; some of these strategies are based on disruption of viral latency, with the goal of inducing cell death or immune-mediated clearance .
Lithium is an inhibitor of GSK-3 (glycogen synthase kinase 3), which induces the intracellular accumulation of β-catenin and the subsequent activation of the TCF/LEF (T-cell factor and lymphoid enhancer binding factor) family of transcription factors. Previous studies reported opposite effects of lithium on HIV-1 replication in vitro[2–4] but, thus far, no data are available about the potential effect of the β-catenin pathway activation on breaking HIV-1 latency in vivo in virologically suppressed patients.
For their efficacy as mood stabilizers, lithium salts have been used to improve the course of bipolar disorder, associated with neuroinflammation and viral persistence in the central nervous system, in viremic HIV-1 infected patients . However, changes in viral load reported in this context can be attributed to a concomitant effect of improved adherence to ART following stabilization of the psychiatric disorder.
Lithium has been recently reported to be also useful for the treatment of HIV-1 associated mild neurocognitive impairment [5,6]. Thus, we based the present investigation on an ongoing study evaluating neurocognitive improvements by lithium administered to HIV-1 infected patients who had been on effective ART for at least 1 year and had no major psychological disorders (TRIANT-TE clinical study; NCT01348282). The Institutional Review Board of the Hospital Germans Trias i Pujol (Badalona, Spain) approved the protocol and all participants provided written informed consent. The present proof-of-concept study, in which nine patients were included, is the first prospective analysis of the effect of lithium on HIV-1 expression and reservoir size in the CD4+ T cells of virologically suppressed patients. Mean time from HIV-1 diagnosis was 10.7 ± 6.5 years, and the mean time of sustained virological suppression was 5.3 ± 3.4 years (Supplementary Table 1, https://links.lww.com/QAD/A543). The initial lithium carbonate dose of 400 mg/day was subsequently titrated to reach blood levels of 0.4–0.8 mmol/l.
Blood samples were collected at baseline and at weeks 2, 4, and 12 to evaluate viral transcription levels. Cell-associated RNA was extracted (RNeasy Mini Kit; Qiagen, Hilden, Germany) from purified CD4+ T cells (CD4 T Cell Isolation Kit; Miltenyi Biotech, Friedrich, Germany) and HIV-1 RNA transcripts were quantified by droplet digital PCR (one-step RT-ddPCR; BioRad Laboratories, Hercules, CA, USA), in parallel with TBP (TATA-box binding protein) as the housekeeping gene . ddPCR was also used to quantify the copy number of HIV DNA and the RPP30 cellular gene, from CD4+ T cell lysed extracts, to estimate the size of the proviral reservoir [7,8]. To evaluate viral load down to 1.3 copies/ml, 5 ml of plasma samples were ultracentrifuged before quantification of HIV-1 RNA using the Cobas Ampliprep/Cobas Taqman HIV-1 Test, version 2.0 (Roche Diagnostics, Manheim, Germany). A limiting dilution virus culture assay was used to measure the replication-competent reservoir in purified CD4+ T cells as previously described  with minor modifications: activation of donor blasts by the ‘3 × 3’ method . Frequency of infectious HIV-1 in the supernatants was determined after 14 days, by TZM-bl culture infection, and calculated using a maximum likelihood method based on a modified algorithm (http://http://www.danielrosenbloom.com/iupmstats).
Contrary to what was expected, 2 weeks after initiation of lithium therapy, cell-associated HIV-1 RNA transcripts decreased in five out of six patients (Fig. 1a). Indeed, a 40% reduction in viral transcription levels was observed at week 4. Of note, viral transcription later increased (P = 0.03), and all patients had recovered their initial transcription pattern after 12 weeks of treatment.
Most patients showed detectable viremia at study entry (67%), but this proportion decreased to 44% immediately after initiation of lithium (Fig. 1b), before rising again to 87% at week 12. Despite the fact that these data are not statistically significant, they paralleled the dynamics of HIV-1 expression in circulating CD4+ T cells.
To further evaluate the effect of lithium on the size and dynamics of the latent viral reservoir, we measured the proportion of circulating CD4+ T cells harbouring proviral DNA. At initiation, we detected a median of 1173 HIV-1 copies per million CD4+ T cells (interquartile range: 388–2343); however, this value decreased to 582 copies (373–1606) after 4 weeks of treatment (Fig. 1c). Longitudinal analysis revealed a statistically significant drop in the size of the proviral reservoir in CD4+ T cells at this point (19% median decrease; P = 0.03). Nevertheless, this effect was not permanent, as longitudinal changes in total HIV-1 DNA were lost at week 12.
The replicative-competent reservoir, measured as the frequency of infectious units per million CD4+ T cells (IUPM) at baseline and week 12, showed a decreasing tendency in most patients (four out of six; Fig. 1d), but these changes did not reach statistical significance, neither at individual level nor overall.
Our results indicate that, upon β-catenin signalling activation induced by lithium treatment, transcription factors with repressor activity might mediate the inhibition of residual viral production in CD4+ T cells from virologically suppressed patients. In the case of memory CD4+ T cells, which harbour most of HIV-1 DNA and mRNA detected in blood of ART-suppressed patients [11,12], β-catenin pathway has been described to be mechanistically related to self-renewal and survival, while activation seems to be suppressed . Further studies might assess the potential involvement of TCF/LEF transcription factors in the establishment of HIV-1-latency in these cells.
The lithium-mediated suppression of viral expression also led to a transient decrease in proviral reservoir, suggesting a potential role for low-level viral production in supporting de-novo infections and continuous replenishment of the viral reservoir during ART. Thus, lithium could alternatively be considered as a potential therapeutic agent aimed at inhibiting transcription from latently infected CD4+ T cells. Nevertheless, this repressor effect was not lasting. As in the case of other agents that also transiently modulate HIV-1 expression, future studies on improved administration strategies (e.g. intermittent doses or combination therapies) [14,15] are necessary to evaluate whether the unresponsiveness of target cells can be overcome, thus enhancing the potential of lithium to reduce the viral reservoir in the long term.
M.C.P. and M.S. performed data acquisition, analysis and interpretation, and wrote the manuscript; S.M.-L. contributed to acquisition and analysis of data; D.O. performed statistical data analysis; J.A.M.-M., J.M. and B.C. contributed to clinical study design and performance, and revised the manuscript; J.M.-P. participated in the study design and data interpretation and wrote the manuscript. Maria C. Puertas and Maria Salgado contributed equally to this work.
This study was supported by the Spanish Ministry of Health and Social Policy under the project ‘The TRI-ANTiretroviral and coadjunctive ThErapies for HIV-associated neurocognitive impairment study (TRIANT-TE)’ [EC10-320], ‘Lluita Contra la Sida’ Foundation, and the Spanish AIDS network ‘Red Temática Cooperativa de Investigación en SIDA’ [RD12/0017/0002]. M.S. was supported by the Sara Borrell programme from the Spanish Health Ministry (CD11/00286). S.M-L. was supported by Agència de Gestió d’Ajuts Universitaris i de Recerca from Generalitat de Catalunya (2013FI_B 00275).
Conflicts of interest
There are no conflicts of interest.
1. Katlama C, Deeks SG, Autran B, Martinez-Picado J, van Lunzen J, Rouzioux C, et al. Barriers to a cure for HIV: new ways to target and eradicate HIV-1 reservoirs
2. Kumar A, Zloza A, Moon RT, Watts J, Tenorio AR, Al-Harthi L. Active beta-catenin signaling is an inhibitory pathway for human immunodeficiency virus replication in peripheral blood mononuclear cells
. J Virol
3. Henderson LJ, Narasipura SD, Adarichev V, Kashanchi F, Al-Harthi L. Identification of novel T cell factor 4 (TCF-4) binding sites on the HIV long terminal repeat which associate with TCF-4, beta-catenin, and SMAR1 to repress HIV transcription
. J Virol
4. Rafati H, LeMasters E, Lennert VDD, El-Sayyed M, Boucher C, Vries R, et al. Activation of the Wnt pathway by natural ligands or small molecule inhibitors activates latent HIV. 7th IAS Conference on HIV Pathogenesis, Treatment and Prevention; 30 June to 3 July 2013; Kuala Lumpur, Malaysia.
5. Letendre SL, Woods SP, Ellis RJ, Atkinson JH, Masliah E, van den Brande G, et al. Lithium improves HIV-associated neurocognitive impairment
6. Schifitto G, Zhong J, Gill D, Peterson DR, Gaugh MD, Zhu T, et al. Lithium therapy for human immunodeficiency virus type 1-associated neurocognitive impairment
. J Neurovirol
7. Archin NM, Liberty AL, Kashuba AD, Choudhary SK, Kuruc JD, Crooks AM, et al. Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy
8. Buzon MJ, Massanella M, Llibre JM, Esteve A, Dahl V, Puertas MC, et al. HIV-1 replication and immune dynamics are affected by raltegravir intensification of HAART-suppressed subjects
. Nat Med
9. Siliciano JD, Siliciano RF. Enhanced culture assay for detection and quantitation of latently infected, resting CD4+ T-cells carrying replication-competent virus in HIV-1-infected individuals
. Methods Mol Biol
10. Prado JG, Prendergast A, Thobakgale C, Molina C, Tudor-Williams G, Ndung’u T, et al. Replicative capacity of human immunodeficiency virus type 1 transmitted from mother to child is associated with pediatric disease progression rate
. J Virol
11. Chomont N, El-Far M, Ancuta P, Trautmann L, Procopio FA, Yassine-Diab B, et al. HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation
. Nat Med
12. Yukl SA, Shergill AK, Ho T, Killian M, Girling V, Epling L, et al. The distribution of HIV DNA and RNA in cell subsets differs in gut and blood of HIV-positive patients on ART: implications for viral persistence
. J Infect Dis
13. Kulpa DA, Brehm JH, Fromentin R, Cooper A, Cooper C, Ahlers J, et al. The immunological synapse: the gateway to the HIV reservoir
. Immunol Rev
14. Rasmussen T, Tolstrup M, Brinkmann C, Olesen R, Erikstrup C, Solomon A, et al. Cyclic dosing of panobinostat to reverse HIV-latency: findings from a clinical trial. 6th International Workshop on HIV Persistence During Therapy. 3–6 December 2013, Miami, FL, USA. IHL Press; 2013. 57.
15. Archin NM, Bateson R, Tripathy MK, Crooks AM, Yang KH, Dahl NP, et al. HIV-1 expression within resting CD4+ T cells after multiple doses of vorinostat
. J Infect Dis
2014; [Epub ahead of print].