Understanding the molecular virology of HIV infection has not only been essential for the development of antiretroviral drugs that block HIV replication but will also be crucial in the development of strategies for curing HIV infection.
Present limitations on therapeutic driven cures
The persistence of HIV infection in the face of antiretroviral therapy (ART) reflects the complex nature of the target cell population - our immune system. The immune system relies on the persistence of long-lived memory CD4+ T cells that can be activated upon future exposure to pathogens. HIV can establish a reservoir of latent infection within this memory CD4+ T cell pool and thus persist for long periods of time (estimated to be several decades with current half-life estimates of 44 months [141, 142]). Treatment with ART is successful in reducing HIV viral loads, reducing onward transmission and limiting viral evolution to a point where it can no longer be detected [143, 144]. However, given the long half-life of the latent reservoir and that it may be clonally expanding over time [145-148], ART needs to be life-long. Indeed, once on ART, it is recommended that patients do not stop therapy, as clinical trials of treatment interruptions have observed recrudescence of HIV replication after ceasing ART [149].
Documented precedents for a HIV cure
The latent HIV reservoir provides the primary challenge towards a HIV cure. In hepatitis C virus (HCV) infection, latent reservoirs are not formed and therapeutic cures can be achieved within only 12 weeks of antiviral therapy [150]. Thus, targeting the latent HIV reservoir has received significant attention in efforts towards a HIV cure. In discussing requirements for a HIV cure, two levels of cure have been considered. Firstly, a sterilising cure, which refers to the eradication of HIV from all cells and body tissues, analogous to HCV cures achieved by antiviral therapy. Determination if a sterilizing cure has actually been achieved will, however, be a challenge. The second type of HIV cure is referred to as a functional cure, where HIV replication is controlled at levels below detection in the absence of ART. In this context, a small subset of individuals referred to as elite controllers (EC) fit the definition of functional cure, as they exhibit persistent plasma HIV RNA levels of <50 copies/mL without ART [151]. Augmentation of immune responses against HIV by vaccination to produce this outcome would be the obvious path to replicate this setting. The genotypes of individuals who are classified as EC are enriched for various traits, compared to the general population, which provides clues as to what type of immune responses should be augmented. For example, HLA-B*5701 is carried by about 65% of ECs compared to 10% of the US caucasian population, highlighting the importance of CD8+ T cell driven viral control in this unique population [152].
Only one case of a HIV cure has been documented (‘The Berlin Patient’). Recently, another potential cure case (‘The London Patient’) has been reported but is cautiously referred to as a patient in long term remission [153]. Whilst these two cases are of particular importance, it is important to note there have been several so-called ‘HIV near cures’, including the Boston patients and the Mississippi baby. Documentation of failed but ‘near cure’ attempts are invaluable in vivo observations that help on the path towards a HIV cure. The first documented ‘near cures’ were observed in what are referred to as the Boston patients [154]. Both patients received an allogeneic hematopoietic stem cell transplantation (HSCT) for treatment of hematologic malignancies and remained on ART. The processes of conditioning for, and engraftment of, the HSCT was associated with a 3 log drop in HIV viral load. However, ART interruption (often referred to as analytical treatment interruptions in HIV cure research) to determine if a HIV reservoir persisted, led to a rebound of HIV replication after 12 weeks in one patient and 36 weeks in the second patient. Additional HSCT cases to the Boston patients have also documented similar findings and in one patient rebound of HIV replication was delayed to 41 weeks [155]. Therefore, whilst HSCT did not result in successful HIV cures, it did highlight that the process of undergoing a HSCT could significantly impact on the HIV reservoir size, as indicated by the lag in rebound of HIV replication once ART was stopped. However, the potential morbidity and mortality associated with HSCT must be considered and compared to that of successful ART. Therefore, HSCT is currently not an acceptable HIV cure strategy at in HIV patients who do not have hematologic malignancies.
In the case of the ‘Mississippi baby’, the child started ART at 30 hours post-birth [156] with therapy ceased 18 months later. Resumption of HIV replication was not detected until around 27 months post-ART cessation [157]. In this context, while early ART led to long-term suppression of HIV replication, it clearly did not prevent the establishment of a HIV reservoir. This case informed the field that even very small viral reservoirs can persist in the face of ART and, importantly, that the reservoir could recover and expand when ART is ceased.
To date, the only known examples of individuals who are considered to be cured of HIV infection are the ‘Berlin patient’ [158, 159] and the ‘London patient’ [153]. The ‘Berlin patient’ was infected with HIV for over 10 years and underwent treatment for acute myeloid leukemia. Treatment consisted of chemotherapy, anti-thymocyte globulin, total body irradiation and finally a HSCT from a donor who was homozygous for the delta 32 mutation in the gene for CCR5, which leads to lack of CCR5 expression on cells and protection from HIV infection. For close to 10 years off ART, the patient had undetectable HIV using current detection methodology. However, around the 10 year mark, the patient commenced PrEP to prevent future HIV infection (disclosed by the patient at the HIV Gene Therapy conference in Seattle, 2017). The London patient represents are similar clinical case with important modifications to clinical conditioning prior to a HSCT with stem cells from a donor homozygous for the delta 32 CCR5 mutation [153]. In brief, the clinical conditioning for the London patient had greater specificity for targeting the lymphoma and could be used at a reduced intensity. In contrast, conditioning for the Berlin patient was of greater intensity and consisted of combined radiation and chemotherapy treatment. Whilst these cases provide hope for development of a HIV cure strategy, the time between identification of the Berlin patient and the London patient (over 10 years) highlights the rarity of ‘HIV cures’ and the challenges likely to be encountered in implementing this specific approach. That said, as additional cases beyond that of the Berlin and London patient are observed, the clinical protocols for co-treating lymphoma and HIV may lead to greater numbers of patients in co-remission for HIV and lymphoma.
As mentioned above, the process of HSCT in the ‘Boston patients’ reduced the size of the HIV reservoir but failed to eradicate it [160]. Of note, both patients had a HSCT from donors who were genetically permissive to HIV infection (ie. did not carry the CCR5 delta 32 homozygote genotype) [154]. Thus, HSCT may require a donor with a homozygous delta 32 mutation to create not only a reduction in the HIV reservoir size but also engraftment of bone marrow with a population of stem cells from which immune cells develop that are resistant HIV infection. However, the low frequency of donors who are homozygous for the delta 32 mutation in the CCR5 gene [161] compromises the feasibility of the Berlin and London patient approaches. In addition, whilst we always note the successful cases, it is also important to consider the repeat attempts at mimicking a cure in this way that have failed. For example, a HSCT carried out on a patient with leukaemia using stem cells from a donor who was homozygous for the CCR5 gene delta 32 mutation was followed by a rebound infection with CXCR4-tropic HIV post-transplant in addition to relapse of the leukemia [162], both contributing to the patient’s death.
Gene therapy
There are many approaches to using gene therapy in HIV cure research at various stages of development. Given the outcome of the Berlin patient, gene therapy to render cells resistant to HIV infection ex vivo is being considered to not only mimic the stem cells of the donor used for the HSCT in the Berlin patient but also to avoid graft versus host disease (as the genetically manipulated cells are derived from the same patient). Advancements in gene therapy techniques that can render cells resistant to HIV include lowering CCR5 expression levels [163-168], expression of peptides that inhibit the HIV fusion reaction [163, 168, 169] and post-entry manipulation that can transcriptionally silence the virus and thus keep it permanently in a dormant state [170-172]). Treatment may also consist of combination approaches, for example current clinical trials are underway that utilize both reduction of CCR5 expression and use of HIV fusion inhibiting peptides (http://www.calimmune.com/news.php?PK_article=1994). Whilst the latter gene therapies have great potential, delivery of the genetic information to the right cells still provides issues in terms of feasibility. For example, it is notoriously difficult for peripheral blood CD4+ T cells to acquire genetic information using existing transduction techniques [173, 174].
Whilst generation of HIV resistance and silencing of the existing HIV reservoir are two means by which gene therapy may contribute towards a HIV cure, others are now using gene therapy to attack the latent reservoir using genetically modified T cells. Chimeric antigen receptor T cells (CAR T cells) are genetically modified CD4+ and CD8+ T cells in which the T cell receptor has been modified to target various antigens, often by using a single chain antibody fragment from an antibody specific to that antigen. This approach has revolutionized cancer therapy. In particular, previously untreatable B cell malignancies can now be treated with this type of gene therapy (reviewed in depth by June and colleagues [175]). Interestingly, preclinical studies in animal models using this approach to target HIV infection have observed promising results [176]. Furthermore, this approach has increased biomedical knowledge at several levels. Firstly, that effective CD8+ T cell responses can lead to control of HIV replication. Secondly, that unique broadly neutralising antibodies to HIV can be isolated and used to generate panels of chimeric antigen receptors to induce CD8+ T cells to attack any cell type expressing HIV envelope glycoprotiens (reviewed in [177]). Finally, that CD8+ T cells can be modified to target specific cells in which HIV may persist; for example, follicular helper CD4+ T cells, which contain a significant reservoir of HIV infection in lymphoid tissues (reviewed by [178]). Thus, investigators are now encoding CXCR5 (the chemokine receptor that controls trafficking of T cells to germinal centres in lymphoid tissue) alongside a chimeric antigen receptor that recognises and targets HIV-infected cells [179].
Whilst gene therapy has the potential to contribute to a HIV cure, accessibility to this treatment will be the biggest challenge moving forward as the cost to treat each person will be in excess of $500,000. Given the current success of gene therapy for many cancers, increased optimisation of gene therapy protocols and more streamlined clinical efforts will hopefully lead to increased accessibility over time.
Reversal of HIV latency in combination with ART
If ART and immune-based therapies are to exert an effect on the latent HIV reservoir, viral latency should be reversed. Several avenues for waking the latent reservoir have been explored and primarily use what is called latency reversing agents (LRAs). The latent reservoir is well known to ‘wake’ as a result of T cell activation and, therefore, early studies attempted to activate CD4+ T cells. However, along with the activation came various toxicities with limited reservoir reduction [180, 181]. Subsequent attempts to refine latency reversal led to the use of candidate LRAs such as HDAC inhibitors [182-186] and methylation inhibitors [187]. Clinical trials using potent HDAC inhibitors have demonstrated transient increases in cell-associated and plasma HIV-1 RNA consistent with waking of the latent reservoir. However, to date limited reservoir reduction has been reported [182, 183, 185, 186, 188]. Yet it is presently unclear if the lack of reduction in the reservoir size is due to lack of latency reversal or the failure to subsequently deplete infected cells. Key to the latter, is the ability of immune responses, driven primarily by CD8+ T cells or neutralizing antibodies, to target infected cells. Thus, augmentation of T cell and antibody responses by therapeutic vaccines, passive immunization with potent neutralising antibodies, or even delivery of genetically modified CD8+ T cells (CAR T cells) may be key to the elimination of HIV reservoirs after latency reversal.