Drugs (2020) 80:859–868 https://doi.org/10.1007/s40265-020-01322-y REVIEW ARTICLE Novel Approaches Towards a Functional Cure of HIV/AIDS Lucia Bailon1,2 · Beatriz Mothe1,3,4 · Lance Berman5 · Christian Brander3,4,5,6 Published online: 20 May 2020 © The Author(s) 2020, corrected publication May 2020 Abstract Therapeutic approaches towards a functional cure or eradication of HIV have gained renewed momentum upon encouraging data emerging from studies in SIV monkey models and recent results from human clinical studies. However, a multitude of questions remain to be addressed, including how to deal with the latent viral reservoir, how to boost the host immune response to the virus and what the hurdles are to reach relevant viral compartments in the body. Advances have been made especially with regard to identifying agents that can reactivate the latent virus in vivo and boost the cellular and humoral immunity, but it remains largely unclear whether any of these strategies can awaken a sufficiently large fraction of the viral reservoir and whether the boosted immunity can prevent rapid viral replication once antiretroviral treatments are stopped. Key Points 1 Introduction HIV cure remains a scientific challenge to this day. The Human Immunodeficiency Virus 1 (HIV-1) has infected more than 70 million individuals since its entry Viral control without antiretroviral treatment is the main into the human population [1]. Thanks to the development goal of a functional cure for HIV. and increasing availability of effective antiretroviral treat- Combination approaches may be needed for successful ment (ART), the rate of new infections has slowed, and virus control. many million lives have been saved. Although ART has dramatically helped to render HIV infection into a gener- ally well-managed chronic infection, issues related to cost, stigma and toxicity still need to be resolved. In response to these needs, the scientific community has aimed to develop strategies that could provide an “HIV cure”, to alleviate the need for life-long ART treatment, prevent forward infec- tions, and ultimately, to remove the virus from the body. As such, a functional cure of HIV infection on one side and The original article has been updated: Due to first author name update. the complete eradication of the virus from infected indi- viduals on the other, remain ambitious goals on the path * Christian Brander towards controlling and ending the HIV pandemic. Progress cbrander@aelixtherapeutics.com has been made towards both, achieving a “functional cure” 1 Fundació Lluita contra la Sida, Infectious Disease (defined as sustained virus control to undetectable/low levels Department, Hospital Universitari Germans Trias i Pujol, in the absence of antiretroviral treatment) as well “sterilis- Badalona, Spain ing cure”, indicating the complete eradication of the virus 2 Universitat Autònoma de Barcelona, Bellaterra, Spain from its latent reservoir hiding places and from the infected 3 IrsiCaixa AIDS Research Institute-HIVACAT​, Badalona, individual. Although no effective and scalable strategy for Spain either goal exists currently, there is scientific evidence that a 4 Faculty of Medicine, Universitat de Vic-Central de Catalunya functional cure and viral reservoir eradication could indeed (UVic-UCC), Vic, Spain be achieved. In fact, 10 years after the "Berlin patient" was 5 AELIX Therapeutics, Barcelona, Spain first reported to be cured of HIV [2], a second individual 6 known as the “London patient” has also been found to be ICREA, Pg. Lluis Companys 23, Barcelona, Spain Vol.:(0123456789) 860 L. Bailon et al. free of any detectable traces of HIV for almost 2 years [3]. CD4+ lymphocytes, which have HIV genomes integrated In these two individuals, both suffering from a haematologi- into the cellular DNA [9]. This latent state is maintained by cal malignancy, HIV cure was achieved after a complex and different mechanisms including the production of low levels risky procedure, including an allogenic stem-cell transplan- of Nf-kB and NFAT, which hinder transcriptional activity, tation from healthy donors who were homozygous for the as well as the presence of immune receptors such as PD-1, CCR5-Δ32 deletion. However, such interventions are asso- CTLA-4 and others, that limit T-cell activation, and thereby, ciated with high morbidity and mortality rates. Thus, they prevent viral reactivation and effective T-cell-mediated kill- are only justified in select individuals with haematologic ing [10]. Furthermore, homeostatic proliferation of latently disorders requiring stem-cell transplant and make them non- infected cells may also be a major driver of the persistence scalable for all HIV-infected individuals. Still, it is impor- of HIV DNA [11]. The viral reservoir is also thought to be tant that the observations with the Berlin patient could be sustained through ongoing residual viral replication, even in repeated and that ongoing studies include a growing number the presence of ART and despite appropriate viral suppres- of individuals to further test these strategies with less inva- sion in the peripheral blood, although this is still a topic of sive and more widely scalable regimens. This will possibly ongoing debate. Replication is further believed to be aided help the field to design more widely applicable approaches by cell-to-cell spread and by insufficient levels of antiretro- including genetic modification “in vivo” and “suppression viral drugs in different tissues and ART sanctuaries [12]. and protection strategies” that are beginning to be tested in Furthermore, aside from T-cell resident viral reservoirs, data pre-clinical models [4]. from humanised mouse models and natural HIV infection indicate that myeloid cells may also contain an important portion of the viral reservoir, even in the brain [13]. This is 2 Viral Reservoir as a Major Obstacle for HIV further evidenced by the recent finding that in individuals Cure who stop their ART, macrophage-tropic HIV-1 variants can be detected in recrudescing viraemia [14]. This clearly war- To date, many HIV cure clinical trials have been conducted rants further research to characterise myeloid reservoirs in in small cohorts, testing different immune-based interven- different tissues, including compartments such as the cen- tions such as therapeutic T cell vaccines, cytokine admin- tral nervous system [15]. Regardless though, since a large istrations, immune checkpoint inhibitors, and a plethora fraction of CD4 + lymphocytes (and other potential reservoir of drugs with variable safety profiles that are more or less cells) are spread throughout different body tissues and only effective in reactivating latent reservoir virus [5]. A major about 2% are found in peripheral blood, it is challenging to challenge for both, functional cure and approaches aiming precisely define the size and most relevant compartments of at total viral eradication, is the existence of a latent viral res- the viral reservoir [16]. ervoir which is formed shortly after acute HIV infection [6]. Aside from uncertainties on the various compartments Over the last years, different aspects surrounding the estab- of the viral reservoirs, advances in the field are also compli- lishment and maintenance of the viral reservoir have been cated by the fact that reservoir measurement and methods intensively studied and mechanisms of viral latency, such are not well standardised, even though in the last decade dif- as long-lived infected cells or residual ongoing replication ferent test and tools have been developed [17]. Among them, have been described. Despite this, many features of the viral the determination of total and integrated HIV DNA by quan- reservoir still remain unknown and pose major challenges titative polymerase chain reaction (PCR) and viral protein for the development of successful HIV curative strategies. quantification (HIV p24) are highly sensitive, even though The latent viral reservoir is established from the first these measures may overestimate the size of the latent res- moments of HIV infection. Animal models have dem- ervoir to some degree [18]. This is due to the fact that most onstrated that within the first 72 h of SIV-infection, viral of the integrated virus in the latent reservoir is replication seeding in lymph nodes and gut-associated lymphoid tis- defective and will not contribute to viral rebound once ART sue is occurring [7]. Recent data from cohorts with acute is stopped [19]. Thus, assays such as the quantitative viral HIV infection support the notion that rapid reservoir seed- outgrowth assay (qVOA) are used to more specifically meas- ing occurs also in humans early after HIV acquisition, even ure the replication-competent reservoir. However, qVOA is under early ART initiation [8]. In addition, as shown in the time intensive and requires large amounts of biological spec- RV254/SEARCH 010 study, if ART treatment is not initi- imen. In addition, qVOA may actually underestimate the size ated early after infection signs of immune damage, gut T-cell of the clinically relevant reservoir due to sampling limita- depletion and gradual increases in reservoir size are known tions of latently infected reservoir cells from the most critical to occur, starting already within first Fiebig stages [8]. Once body compartments and the inability to reactivate all intact established, a large portion if not the vast majority of the virus-carrying cells in vitro [20]. As an alternative to qVOA, latently infected cell population is constituted by memory different assays are now available to determine the inducible Novel Approaches Towards a Functional Cure of HIV/AIDS 861 HIV reservoir and the viral transcriptional activity, includ- the HIV-specific cytotoxic T cell response in natural infec- ing TILDA (Tat/Rev-induced limiting dilution assay) [16], tion have shown that virus-specific CD8 T-cell responses IPDA (intact pro-viral DNA assay) and Quadruplex qPCR critically contribute to virus control and their expansion and (Q4PCR, [18, 21, 22]. In addition, several other innovative re-education by therapeutic vaccination may offer an oppor- strategies, both laboratory- and image- (PAT-based imaging/ tunity to achieve sustained functional cure. This may parallel nuclear medicine) based approaches are being developed to the situation in so-called long-term non-progressors (LTNP) determine the actual size and tissue locations of the latent or “HIV elite controllers” who represent < 0.5–1% of all peo- reservoir at different stages of HIV infection. These efforts ple living with HIV but in whom clear evidence exists that may also help to resolve some of the controversy that exists their HIV infection with an intact, replication-competent concerning the specific markers of latently HIV-1-infected virus can be controlled by adaptive immune mechanisms. cells. For instance, the cell surface protein CD32a has been The characterisation of the immune parameters and the fine postulated to specifically define a cell population enriched in specificity of T-cell responses mediating this control have latently infected cells [23]; however, a series of subsequent been the subject of many studies and have given rise to dif- publications have questioned this finding [24]. ferent immunogen designs that are currently being employed Together, given the uncertainties surrounding the rel- in different therapeutic vaccine strategies [27, 28]. evant characteristics of the latent viral reservoir, especially A similar but mechanistically possible different state of in terms of exact location, size, genetic composition as well viral control is that of post-treatment controllers (PTC). as its mechanisms for maintenance, it may be difficult to These PTCs are HIV-infected individuals who are able to develop targeted interventions that can remove the latent maintain suppressed undetectable or low-level viraemia HIV reservoir at once and completely [25]. As eluded to after ART is stopped but without having received any prior above, to date, only highly invasive strategies such as bone immune therapeutic treatment. The incidence of PTC varies marrow transplants from CCR5-mutated donors have been widely, depending on reports and the definition of sustained successful in removing the entire reservoir. Thus, the persis- control. In the first report of a group of PTCs (n = 14) in a tence of parts of the viral reservoir remains one of the most retrospective cohort [29], PTCs were estimated to be near significant hurdles in developing a cure for HIV infection. 15% of HIV individuals who had received ART early after acquisition. Since then, several studies have reported a large range of PTC incidence. The most in-depth meta-analysis of 3 Models of “Functional” Cure PTC was conducted in the CHAMP (Control of HIV after antiretroviral Medication Pause) study, which included more As eluded to above, HIV cure can be an ambiguous term than 700 HIV-infected individuals who discontinued ART that is often used to refer either to a “sterilising cure” mean- in 14 selected interruption trials [30]. The investigators ing the eradication of the virus from the infected individual reported PTC rates of up to 13% in early treated individuals or, alternatively, to the induction of a state of “functional and less than 5% in individuals who initiated ART during cure” or ‘ART-free remission’ where pro-viral HIV DNA is chronic stages of infection. Importantly, the CHAMP study still detectable in blood cells but plasma viraemia remains has identified different profiles of viral kinetics leading to undetectable or at very low levels in the absence of ART. PTC, including 31% of PTCs who presented viral rebounds In contrast to eradication, in functional cure the virus is not with peak viraemia up to 10.000 copies/mL before control- being eliminated from the body, a viral reservoir persists ling viral replication spontaneously (post-rebound control- and there remains a risk of late breakthrough of long-term lers). These contrast with the PTCs that are able to maintain controlled infection [26]. Persistent control of viraemia may an undetectable/low-level plasma viral load for months/ be highly depend on effective antiviral immunity, and thera- years after ATI (non-rebounders). Interestingly, PTCs are peutic vaccines may play a critical role in the induction of not enriched for individuals who express certain benefi- such an immune-based “functional” cure. However, thera- cial HLA-I alleles classically associated with HIV control peutic vaccination may face the critical challenge that the (HLA B*57/HLA B*27 and others). Also, PTCs do not show host immune system is at least partly dysfunctional due to particularly high magnitudes of interferon-gamma (IFN-γ) HIV infection, which can drive inflammation and exhaus- secreting, HIV-specific CD8 T-cell responses but seem to tion of the innate and adaptive immune system even with have preserved robust HIV-specific CD4 T-cell immunity ART suppressed viraemia. These detrimental effects are [31]. These observations are of critical importance for the even more pronounced in the absence or delayed initiation of study of immune interventions aiming at functional cure as antiretroviral treatment, where the persistent antigen stimu- the rate of spontaneous control (i.e. PTC) and viral dynamics lation causes extended exhaustion of the virus-specific T cell during treatment interruption needs to be considered when response and/or can lead to a progressive escape of virus measuring effectiveness of functional cure strategies in clini- from T-cell-mediated immune surveillance. Still, studies of cal trials. 862 L. Bailon et al. 4 Measuring Viral Control During Analytical inference from non-human primate (NHP) models, where Treatment Interruptions higher levels of control have been observed and where animals remain off treatment for time intervals that may A major challenge with ethical, medical and public health not be considered appropriate in humans, participants in dimensions arises in the clinical testing of functional cure therapeutic vaccine/functional cure studies should possibly therapies when it comes to how to best measure the effec- remain off ART long enough to observe the kinetics of the tiveness of such interventions. Since there are currently no viral rebound until the ATI set-point is achieved. This will robust virologic or immunologic correlates that can predict certainly not be achieved if the participants are put back viral rebound once ART is stopped, analytical treatment on antiretroviral treatment as soon as viraemia becomes interruption (ATI) is the only hard endpoint by which to detectable. Considering that viral rebound and reservoir assess the effectiveness of functional cure strategies [32]. dynamics after ART cessation show a median rebound Many of the current studies and clinical trials for HIV time of 2 to 4 weeks, latest recommendations suggest a cure (https​://www.treat​menta​ction​g roup​.org/cure/trial​s/) cut-off for ART re-initiation after detecting > 1000 copies/ include analytical treatment interruptions (ATI), but ATI mL for 4 consecutive weeks and up to 100,000 cop/mL is not recommended in routine clinical practice and its in a single determination [36]. Independently from viral implementation is limited to the scope of HIV research and rebound kinetics though, clinical parameters also need to through close clinical monitoring. In addition, potential be considered, especially CD4 counts for which a drop adverse effects of treatment interruption and viral rebound to < 350 cells per µL or CD4% < 15%, dependent on the need to be monitored. For instance, the Strategies for Man- CD4 entry criteria, would prompt re-initiation of ART agement of Antiretroviral Therapy (SMART) study [33] [36]. However, without better defined rebound kinetics, showed that prolonged treatment interruptions—especially it is evidently difficult to establish the timing of the viral on those with low nadir CD4 T-cell counts, increases the rebound after treatment interruption in the context of ther- risk of opportunistic infections or death, highlighting the apeutic vaccination. Of note, the immunological, virologic potential risks of unsupervised treatment interruptions. In and host mechanisms influencing different control profiles addition, presentation of acute retroviral syndrome or viral (for instance PTC, elite controllers, vaccine controllers) rebound in the CNS are other potential complications of are likely to be different from each other, although to date extensive ATI that require close clinical monitoring [34]. these differences have not yet been defined. In addition, On the other hand, short-term ATI as a test to measure the absence of markers of virus control and the recent outcome of therapeutic vaccination have proven to be description of profiles such as post-rebound controllers, relatively safe in terms of development of ART resist- both in the animal model [39] and in early treated HIV ance mutations, reservoir re-seeding, and preservation of infection, requires ATI in clinical trials to be designed HIV-specific immune response, at least when ART was with placebo control groups if a sample size is sufficient restarted within 6 months [19]. In studies with a longer to discern an effect of the intervention. Finally, a major antiretroviral therapy interruption, up to 48 weeks, an challenge for clinical trials that include an ATI arises from increase in reservoir levels measured as integrated HIV- how to manage the risk of HIV transmission to sexual part- 1-DNA was observed, even though total HIV-1-DNA ners, apart from the emotional stress that could manifest returned to the pre-ATI levels within less than six months itself in people with previously undetectable viral load thereafter [35]. for a long time [40, 41]. Added to this is how to manage Recent efforts [36] have tried to establish a consensus the risk of HIV superinfection in individuals off ART [42, on monitoring intervals during ATIs and on virologic and 43]. HIV transmission prevention counselling and the use immunological criteria for ART resumption. The current of pre-exposure prophylaxis (PrEP) in participants’ sexual differences are not only due to preferences by individual partners can actively contribute to preventing new HIV investigators and clinicians, but are also impacted by the infections and reducing the associated stress during the different positions taken by regulatory agencies in differ- ATI period. Together, these considerations highlight that ent countries, complicating the interpretation of data from much work for the trial sponsors and involved physicians different studies. Of critical importance to bear in mind is required and only well-informed, psychologically stable is that stringent viral load thresholds for ART re-start in and highly motivated participants should be included in early-treated individuals may impact the outcome of the interruption trials if one wants to minimise the loss to fol- ATI and may miss potential post-intervention control- low up and some of the outlined complications. lers [37]. In contrast, other studies that have allowed for As described above, it is well established that ART does longer intervals off treatment and for higher viral loads not completely remove HIV from the infected body due to during ATI showed a somewhat better efficacy [35, 38]. By the presence of the latent reservoir, homeostatic proliferation of latently infected cells and virus harbouring in anatomical Novel Approaches Towards a Functional Cure of HIV/AIDS 863 reservoirs not accessible to ART and/or immune surveil- HIV-1 RNA, antibody negative-Fiebig stage I). During the lance. As a consequence, when ART is interrupted, the following 2 years, only low-level HIV RNA was intermit- virus rebounds typically within 4 weeks [44]. Since time to tently detected from different CD4+ T cell subsets from one rebound has been associated with the size of the reservoir, of the participants, despite wide sampling from different tis- several strategies to impact the size of the reservoir have sues (GALT, lymph nodes, bone marrow, CSF, CD4+ T-cell been explored. In particular, the approach of intensified ART subsets and plasma). However, and despite the absence of has widely been researched. However ART treatment inten- detectable HIV, viral rebound was detected 225 days after a sification in long-term suppressed patients, for example with treatment interruption [52]. Likewise, in perinatally infected the integrase inhibitor raltegravir, showed no relevant differ- babies, small HIV reservoir sizes have been directly related ences between the intensified and non-intensified regimen to early ART initiation, promoting a long-term remis- when measuring reservoir levels using ultra-sensitive plasma sion after discontinuation of treatment. Examples such viraemia and integrated and total HIV-DNA determination as the “Mississippi baby” [53], the “French girl” [54] or [45]. Similarly, upon intensifying an integrase-inhibitor- the recently reported “South African boy” [55] show how based regimen with maraviroc, no significant effect was early treatment may limit the size of the viral reservoir and observed in decreasing the reservoir size in early HIV infec- provide these children with advantages in terms of time to tion [46]. On the other hand, in a large, early infection Thai rebound if ART should become interrupted. cohort the “MegaHAART” strategy that combined tenofovir/ Aside from early treatment initiation, the time on ART emtricitabine/efavirenz/raltegravir/maraviroc showed better before treatment interruption may also be critical, as the outcomes in terms of immune function and decreasing reser- reservoir shows a gradual decline under prolonged period voir size compared to individuals who received regular ART of ART. This is illustrated by the SPARTAC study, which (tenofovir/emtricitabine/efavirenz)” [8]. included participants with primary HIV infection who Similarly, early treated individuals showed a more pro- were randomised to either a long (48-week) or short (12- found decrease in cell-associated HIV DNA compared to week) course of ART or no ART at all [56]. In this study, patients treated at chronic stages of HIV infection [47, 48]. control was defined as plasma viral load < 400 copies/mL However, even in these favourable “reservoir conditions” for > 16 weeks. After ATI, the 48-week ART arm showed achieved by very early treatment, a recent clinical trial a higher frequency of post-treatment controllers (18.6%) in showed that individuals initiating treatment in Fiebig stage I contrast to the 12-week ART arm (11.3%) and to the no ART still rebounded quickly after ART cessation [37]. As hypoth- arm (7.9%) [57]. esised by the authors, the immune responses generated dur- ing Fiebig I—which are low compared to responses progres- sively induced in later stages—might be unable to affect 5 Kick‑and‑Kill and Block‑and‑Lock viral rebound but could contribute to reduced viraemia. Of Strategies importance to note are the conservative ART resumption criteria which were used in the RV411 study and which may Described by Siliciano and Deeks in 2012, “Kick and kill” have blunted the possibility to detect post-treatment con- strategies are combined interventions that are based on the trollers after an initial viral rebound, as observed in other reactivation of latently infected cells through latency revers- studies in which PTC have been described [30, 49]. Still, ing agents (LRA) and an immunotherapeutic intervention early treatment in the first six months after HIV acquisi- (such as a therapeutic T-cell vaccine), that lead to the elimi- tion may help to preserve the functionality of HIV-specific nation of cells with reactivated virus [58, 59]. Since neither T-cell-immune response, limit the evolution of the virus and component when tested alone may show clinical benefit, the number of immune escape mutations, reduce immune combination strategies may need to be tested. In addition, activation/exhaustion marker levels, and enhance immune many proposed LRAs may have considerable safety risks recovery in comparison with non-early treatment [50, 51]. and/or are only modestly effective in reactivating latent cells Thus, even though early treatment per-se may not prevent in vivo. In fact, as different mechanisms are involved in the rapid HIV rebound once ART is stopped, the lower reser- maintenance of latency and various cell compartments con- voir levels achieved and the improved immunologic and tribute to the total viral reservoir, a single LRA may not be virologic parameters may provide critical advantages to sufficiently effective and the complete reactivation of the early-treated individuals making them ideal candidates for entire latent viral reservoir may be a major hurdle for effec- early-stage trials researching a functional cure [37, 50]. This tive curative strategies [25]. view is further supported by indirect evidence from indi- Different classes of LRAs have been and are currently viduals recruited from a pre-exposure prophylaxis program being developed and tested clinically, including epigenetic (PrEP) who started prophylactic tenofovir/emtricitabine an modifiers, protein kinase C agonists, NF-κB agonists, Pi3K/ estimated 10 and 12 days after infection (detectable plasma Akt pathway activators, toll like receptor (TLR) agonists, 864 L. Bailon et al. immune checkpoint blockers and other small molecules with but may well serve as future agents that could be explored different mechanisms and potential to disrupt viral latency for their transcriptional blocking strategy [71, 72]. [60]. Of these approaches, the use of Histone Deacetylase Inhibitors (HDACi) has been widely explored. During viral latency, histones are deacetylated, leading to highly com- 6 Combination Strategies Using T‑Cell pacted chromatin, which hinders (viral) gene expression. Vaccines, Passive Nab Immunisation Inhibition of these histone deacetylases thus enables the and Immune Check Point Inhibitors unpacking of chromatin and transcription of viral genes, rendering the infected cells susceptible to immune recogni- Therapeutic vaccines may prove critical in “kick and kill” tion by the cellular arm of the immune response. Initially and functional cure strategies as they may be needed to developed in the field of oncological therapies, the HDA- remove reactivated virally infected cells as soon as they Cis vorinostat, panobinostat and romidepsin have been become visible to the immune system. By inference from shown to reactivate HIV in vitro and in vivo [61]. The first natural HIV infection and studies in HIV controllers who in-vivo proof-of-concept with vorinostat [suberoylanilide do not take ART, increasing the breadth and the magnitude hydroxamic acid (SAHA)] in chronically HIV-infected indi- of the cellular immune responses towards selected and the viduals on suppressive ART, showed an increase in HIV gag most vulnerable targets of the virus appears to be a central gene transcription measured by HIV RNA expression in rest- pre-requisite for successful cure approaches [73]. Different ing CD4 cells [62]. Similarly, panobinostat, a non-selective strategies to induce such responses have been developed, HDACi (pan-HDACi), demonstrated in vitro and in vivo and immunogen sequence design and vaccine vector choices activity in a clinical trial with 15 aviraemic patients, increas- can differ significantly. In addition, most potent vaccination ing cell-associated HIV-RNA and allowing for the detection strategies may need to include elaborate prime-boost regi- of HIV RNA in the peripheral blood [63]. Romidepsin is men, adding further complexity and costs to the production the HDACi that has shown the highest activity on histone and clinical testing of promising strategies [74]. acetylation and HIV replication in several in vitro studies. Aside from vectored vaccines, cell-based approaches for However, as for panobinostat or vorinostat, no changes in immune restoration have also been explored in HIV cure reservoir size measured by total HIV-DNA, Tat/rev Induced strategies. Of special interest in this context have been Limiting Dilution Assay (TILDA) or infectious units per mil- dendritic cells, which as professional antigen present cells lion (IUMP) were observed [64]. Still, HDACis have been (APC) play an important role the induction of both the tested in interventions that include a treatment interruption humoral as well as cellular adaptive immunity. Different period to assess in vivo efficacy to prevent viral rebound. studies, among them the DCV-2 trials [75], provided proof Despite some promising results from the recent open-label of concept that using autologous monocyte-derived den- single arm BCN02 study in early-treated HIV-1-infected dritic cells pulsed with inactivated virus can induce broad individuals using MVA.HIVconsv vaccine [65], these mol- virus-specific T-cell immune responses with the potential to ecules used as single LRA are likely not sufficiently potent impact viral setpoint during an ART interruption phase [38]. to reactivate a substantial proportion of the latent reservoir Similarly, the AGS-004 Phase IIb trial in early-treated HIV- and reduce the overall reservoir size. In addition, in vitro infected individuals employed a regimen of autologous DC studies and data emerging from the BCN02 trial strongly electroporated with RNA encoding autologous viral antigens suggest that romidepsin and possible other HDACi as well, and showed an increase in memory T-cell responses, which may impair host immunity [66, 67], further limiting the use however, was not able to delay viral rebound during ATI of this class of LRAs in HIV cure strategies. [76]. Aside from the above-mentioned interventions that aim to Of note, immunotherapies that are not based on active reverse viral latency and activate viral gene expression, there vaccination strategies, have also been tested in the HIV cure are strategies that aim to achieve the opposite. These “Block- field. In particular, immune therapeutic interventions using and-Lock” strategies aim to lock HIV into a non- or hard-to- broadly neutralising antibodies (bNAb) have shown very reverse latent form and thereby reduce the chance of viral encouraging results. Such bNAbs can, aside from direct neu- reactivation and replication. To date, multiple targets and tralisation of the virus, mediate various effector functions drugs have been postulated to serve as such latency-promot- including ADCC (antibody-dependent cellular toxicity), ing agents (LPA). In particular, molecules that inhibit clas- ADCP (antibody-dependent cellular phagocytosis), ADCDC sical transcriptional factors such as NF-κB, NFAT (CsA), (antibody-dependent complement-dependent cytotoxicity) P-TEFb complex, mTOR complex and Transcription Factor or ADCT (antibody-dependent trogocytosis), which may II H (TFIIH) as well as other drugs such as levosimendan contribute to variable levels of HIV control [77–79]. These [68], spironolactone [69] and complex-targeting curaxin effects may be further increased, and viral escape largely CBL0100 [70] have been tested outside of HIV infection prevented by the use of combination of various bNAbs. Novel Approaches Towards a Functional Cure of HIV/AIDS 865 Indeed, the successful combinations of three bNAbs in pre- treatments for malignancies, their potential as HIV viral res- clinical phases [80, 81] prompted the strategy to be tested ervoir activators can be assessed. Of interest, recent results in HIV-infected individuals, which has shown promise in in a French study showed a significant decrease of the viral a recent a Phase I clinical trial that combined the bNAb reservoir during the treatment of a non-small-cell lung can- 3BNC117 and 10–1074 (NCT03571204). In this trial, effec- cer with nivolumab in an individual patient [91]. However, tive suppression of viral rebound for a median of 21 weeks this effect was not observed in a subsequent small sample was observed, further raising hopes that such interventions of patients (n = 3) treated with anti PD-1 in a similar malig- alone or in combination with T-cell vaccines could pro- nancy [92]. Ongoing studies (among them NCT02408861, vide significantly prolonged virus control [82]. The clini- NCT02595866) that are enrolling a larger number of patients cal benefit of such bNAb-based interventions may be fur- receiving different anti-PD1 treatments for the treatment of ther increased by targeted antibody engineering that allows neoplasia may help to better assess the potential of these the potentiation of specific effector functions (for instance agents in the cure area. through modifications in the Fc portion of the Ab), and the creation of multi-specific antibodies, which might enhance the possible vaccinal-effect of antibodies. This could drive T-cell immunity [83, 84] and possibly, most importantly, 7 Conclusions increase the in vivo half-life of these Abs [85]. Together with increased half-lives, the “vaccinal effect”, whereby the for- In conclusion, much progress has been made over the last mation of immune complexes of reactivated virus and nAb few years in designing and testing different strategies to is believed to boost virus-specific T-cell immunity and effec- achieve a functional cure and eradication of HIV. To date, no tive CTL responses [86], could provide critical advances to scalable therapeutic interventions exist that provide lasting combination approaches. Additional ways to harness the full suppression of viral replication in the absence of antiretrovi- capacities of the humoral immune response and bNAb for ral treatment. Therapeutic vaccines, the passive administra- HIV control is their combination with LRAs and immune tion of bNAb, latency-reversing agents, immune adjuvant as modulators. In this regard, a recent study by Borducchi et al. well as immune checkpoint modulators have all been tested [39] demonstrated that the combination of the TLR7 agonist individually or in combination in NHP models and in human GS9620 (vesatolimod) together with the bNAb PGT121 was clinical trials. Since each individual approach has its specific able to delay SIV rebound for more than 6 months. Mecha- shortcomings, the clinical effectiveness of a future approach nistically, GS-9620 may have activated parts of the latent will likely only be established when all the required compo- viral reservoir, which in the presence of PGT121 led to nents are combined in the right order, schedule and dose, and virus neutralisation. Other TLR ligands, in particular TLR9 in the most suitable patent population, making the clinical agonist (MGN1703, lefitolimod) have been tested clinically development of such strategies a formidable challenge. and may show some effect on viral reservoir as well as the virus-specific T-cell response [87]. However, the effect on Compliance with Ethical Standards reservoir reactivation needs to be assessed and interpreted The present work was funded in parts by the European Union’s Hori- critically, as none of the existing data suggest a broad reac- zon 2020 research and innovation programme under grant agreement tivation of latent virus in vivo. 681137-EAVI2020 and by NIH Grant P01-AI131568. BM is a consult- Finally, alternative strategies to potentiate T-cell vaccine ant for AELIX Therapeutics, S.L., outside the submitted work. CB is have been explored, including the use of immune check- CSO and shareholder. AELIX Therapeutics, S.L., outside the submitted work. LBe is former CMO of AELIX Therapeutics, S.L. LBa declares point inhibitors (ICB). The rational for these strategies is no conflicts of interest. based on the observation that HIV-specific CD8 T cells express high levels of PD1 [88] and that anti-PD1 mAb and Open Access This article is licensed under a Creative Commons Attri- other immune checkpoint inhibitors (CTLA-4, LAG-3, TIM- bution-NonCommercial 4.0 International License, which permits any 3, TIGIT y 2B4) have shown promise in the cancer field to non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the restore anti-cancer immunity. These approaches are currently original author(s) and the source, provide a link to the Creative Com- being explored in the HIV field, in some cases in combina- mons licence, and indicate if changes were made. The images or other tion with therapeutic vaccination and a LRA. In addition, third party material in this article are included in the article’s Creative PD1 is believed to play an important role in the establish- Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons ment and maintenance of latently infected cells [89] and licence and your intended use is not permitted by statutory regula- PD1-blocking treatment has been proposed to increase HIV tion or exceeds the permitted use, you will need to obtain permission DNA transcription [90]. Since monoclonal antibodies block- directly from the copyright holder. To view a copy of this licence, visit ing PD-1 (nivolumab and pembrolizumab) and CTL4 (ipili- http://creat​iveco​mmons​.org/licen​ses/by-nc/4.0/. mumab) are now being used in HIV-infected individuals as 866 L. Bailon et al. References 20. Olvera A, Martinez JP, Casadellà M, Llano A, Rosás M, Mothe B, et al. 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