Broad CTL response is required to clear latent HIV-1 due to dominance of escape mutations

Can HIV Be Cured and Should We Try?

In spite of prolonged and intensive treatment with combined antiretroviral therapy (cART), which efficiently suppresses plasma viremia, the integrated provirus of HIV-1 persists in resting memory CD4+ T cells as latent infection. Treatment with cART does not substantially reduce the burden of latent infection. Once cART is ceased, HIV-1 replication recrudesces from these reservoirs in the overwhelming majority of patients. There is increasing evidence supporting a role for noncoding RNAs (ncRNA), including microRNAs (miRNAs), antisense (as)RNAs, and short interfering (si)RNA in the regulation of HIV-1 transcription. This appears to be mediated by interaction with the HIV-1 promoter region. Viral miRNAs have the potential to act as positive or negative regulators of HIV transcription. Moreover, inhibition of virally encoded long-asRNA can induce positive transcriptional regulation, while antisense strands of siRNA targeting the NF-κB region suppress viral transcription. An in-depth understanding of the interaction between ncRNAs and the HIV-1 U3 promoter region may lead to new approaches for the control of HIV reservoirs. This review focuses on promoter associated ncRNAs, with particular emphasis on their role in determining whether HIV-1 establishes active or latent infection.

Early and late cytotoxic T lymphocyte responses in HIV infection

HIV-1 establishes a lifelong infection in the human body, but host factors that influence viral persistence remain poorly understood. Cell-intrinsic characteristics of CD4 T cells, the main target cells for HIV-1, may affect the composition of the latent viral reservoir by altering the susceptibility to CD8 T-cell-mediated killing. We observed that susceptibilities of CD4 T cells to CD8 T-cell-mediated killing, as determined in direct ex vivo assays, were significantly higher in persons with natural control of HIV-1 (elite controllers) than in individuals effectively treated with antiretroviral therapy. These differences were most pronounced in naive and in terminally differentiated CD4 T cells and corresponded to a reduced viral reservoir size in elite controllers. Interestingly, the highest susceptibility to CD8 T-cell-mediated killing and lowest reservoirs of cell-associated HIV-1 DNA was consistently observed in elite controllers expressing the protective HLA class I allele B57. These data suggest that the functional responsiveness of host CD4 T cells to cytotoxic effects of HIV-1-specific CD8 T cells can contribute to shaping the structure and composition of the latently infected CD4 T-cell pool.

Reactivation of Latent HIV-1 by Inhibition of BRD4

Specific targeting of HIV infected cells has been a challenge since the discovery of the virus. To date, antiretroviral therapy is capable of inhibition of viral spread and controlling the viral load at levels below detection limit. However, the latent viral reservoir persists even during intensive treatment, and produces new infectious viral particles as soon as medication is discontinued. But antiretroviral therapy is cost-intensive, causes major side effects and involves close monitoring of the patient. Relapse due to viral mutation may also require multiple changes in the medication strategy. It is therefore essential for a sustainable cure, to also fully eliminate the latent viral reservoir. Chimeric Antigen Receptor (CAR)-T cell therapy has been very suc-cessful in the treatment of hematologic malignancies. Autologous T cells of patients are genetically modified to express a chimeric receptor, which enables them to specifically bind their tar-get antigen and deplete the target cell without the need of MHC-presentation. Transferring this CAR-T cell technology from targeting of malignant B cells to HIV-infected T cells could potentially be the way to a sterilising cure. However, the development of anti-HIV CAR-T cells has been dormant in the past years due to lack of efficiency, and because targeting of viral proteins on the surface of infected cells did not deplete the latent viral reservoir, hence did not show any improvement compared to conventional antiretroviral therapy. Fast progress in recent development of next generation CARs led to significant enhancement of efficacy and specificity. In addition to that, targeting of cellular rather than viral proteins could potentially overcome persistence of the latent reservoir. Therefore, CD4 as the exclusive HIV entry receptor into the host cell could be a suitable cellular target for anti-HIV CAR-T cells, because all infected cells express CD4, including the latent reservoir. One aim of this thesis was to investigate whether CAR-T cells can specifically and efficiently deplete their CD4-positive target cell population. Therefore, a second-generation CAR was utilised, bearing CD3zeta and CD28 co-stimulatory domains, as successfully used in clinical trials. A CD4-specific Designed Ankyrin Repeat Protein (DARPin) was used as a binding domain, since this molecule was shown to be small, well expressed on the cell surface, specific and not immunogenic. Generation of an expression plasmid, encoding all CAR domains in a cassette system, provided a platform for flexible and individual CAR design. Transduction of primary human T cells with gamma-retroviral particles led to CAR expression on the cell surface. Co-culture resulted in dose-dependent CAR-mediated T cell activation and cytotoxicity towards target but not towards non-target cells, verifying specificity and potency of anti-CD4-DARPin CAR-T cells. High efficiency was shown during co-culture with non-target cells including de-creasing amounts of target cells. CD4 specific cytotoxicity was also observed against very low target cell levels. Human CD4 was selected as a target because it is the entry receptor for HIV into the host cell, and therefore expressed on all HIV-positive cells, irrespectively of their activation state. Another question of this thesis was therefore, if CD4-specific CAR-T cells have the potential to deplete the CD4-positive T cell compartment and thereby also the latent HIV reservoir. Anti-CD4-DARPin CAR-T cells showed efficient and specific cytotoxicity against an HIV-latency model - towards activated, as well as latently infected cells with low levels of target expression. Furthermore, CD4 T cells were efficiently depleted in an autologous setup, without major impact on the CD4-negative T cell population. Taken together, anti-CD4-DARPin CAR-T cells have shown specificity, efficiency and potency, the main properties, required for success-ful CAR-T cell therapy. With regard to further investigation of this approach in an immunocompetent animal model, anti-CD4-DARPin CARs were not only expressed on human, but also non-human primate T cells. Moderate depletion of autologous CD4 target cells was observed using T cells of rhesus macaques, providing the groundwork to test this approach in a well-established immunocompetent animal model. In summary, the results of this work provide evidence, that anti-CD4-DARPin CAR-T cells are a promising approach to specifically deplete potential HIV host cells, along with elimination of latently infected cells. This was achieved by combination of a potent 2nd generation CAR, driving efficient T cell activation with a specific and well expressed DARPin binding domain, target-ing a cellular protein to capture all potentially HIV-positive cells, including those in a latent stage. These data provide a basis for further investigation of anti-CD4-DARPin CAR-T cells for HIV therapy.

HIV-1 persists in a latent reservoir despite antiretroviral therapy (ART). This reservoir is the major barrier to HIV-1 eradication. Current approaches to purging the latent reservoir involve pharmacologic induction of HIV-1 transcription and subsequent killing of infected cells by cytolytic T lymphocytes (CTLs) or viral cytopathic effects. Agents that reverse latency without activating T cells have been identified using in vitro models of latency. However, their effects on latently infected cells from infected individuals remain largely unknown. Using a new ex vivo assay, we demonstrate that none of the latency-reversing agents (LRAs) tested induced outgrowth of HIV-1 from the latent reservoir of patients on ART. Using a quantitative reverse transcription PCR assay specific for all HIV-1 mRNAs, we demonstrate that LRAs that do not cause T cell activation do not induce substantial increases in intracellular HIV-1 mRNA in patient cells; only the protein kinase C agonist bryostatin-1 caused significant increases. These findings demonstrate that current in vitro models do not fully recapitulate mechanisms governing HIV-1 latency in vivo. Further, our data indicate that non-activating LRAs are unlikely to drive the elimination of the latent reservoir in vivo when administered individually.

Reactivation of latent HIV-1 is a necessary step for the purging of the viral reservoir, although it does not seem to be enough. The stimulation of HIV-1 specific cytotoxic T lymphocytes (CTL) may be just as essential for this purpose. In this study, we aimed to show the effect of galectin-9 (Gal-9), known to revert HIV-1 latency, in combination with the blockade of TIM-3, a natural receptor for Gal-9 and an exhaustion marker. We confirmed the ability of Gal-9 to reactivate latent HIV-1 in Jurkat-LAT-GFP cells, as well as in an IL-7-based cellular model. This reactivation was not mediated via the TIM-3 receptor, but rather by the recognition of the Gal-9 of a specific oligosaccharide pattern of resting memory CD4+ T cells' surfaces. The potency of Gal-9 in inducing transcription of latent HIV-1 was equal to or greater than that of other latency-reversing agents (LRA). Furthermore, the combination of Gal-9 with other LRA did not show synergistic effects in the reactivation of the latent virus. To evaluate the impact of TIM-3 inhibition on the CTL-response, different co-culture experiments with CD4+T, CD8+ T, and NK cells were performed. Our data showed that blocking TIM-3 was associated with control of viral replication in both in vitro and ex vivo models in cells from PLWH on antiretroviral therapy. A joint strategy of the use of Gal-9 to reactivate latent HIV-1 and the inhibition of TIM-3 to enhance the HIV-1 CTL specific-response was associated with control of the replication of the virus that was being reactivated, thus potentially contributing to the elimination of the viral reservoir. Our results place this strategy as a promising approach to be tested in future studies. Reactivation of latent-HIV-1 by Gal-9 and reinvigoration of CD8+ T cells by TIM-3 blockade could be used separately or in combination.ImportanceHIV-1 infection is a health problem of enormous importance that still causes significant mortality. Antiretroviral treatment (ART) has demonstrated efficacy in the control of HIV-1 replication, decreasing the morbidity and mortality of the infection, but it cannot eradicate the virus. In our work, we tested a protein, galectin-9 (Gal-9), an HIV-1 latency-reversing agent, using an in vitro cellular model of latency and in cells from people living with HIV-1 (PLWH) on antiretroviral therapy. Our results confirmed the potential role of Gal-9 as a molecule with a potent HIV-1 reactivation capacity. More importantly, using a monoclonal antibody against T cell immunoglobulin and the mucin domain-containing molecule 3 (TIM-3) receptor we were able to enhance the HIV-1 cytotoxic T lymphocytes (CTL) specific response to eliminate the CD4+ T cells in which the virus had been reactivated. When used together, i.e., Gal-9 and TIM-3 blockade, control of the replication of HIV-1 was observed, suggesting a decrease in the cellular reservoir.

Current antiretroviral therapy (ART) provides potent suppression of HIV-1 replication. However, ART does not target latent viral reservoirs, so persistent infection remains a challenge. Small molecules with pharmacological properties that allow them to reach and activate viral reservoirs could potentially be utilized to eliminate the latent arm of the infection when used in combination with ART. Here we describe a cell-based system modeling HIV-1 latency that was utilized in a high-throughput screen to identify small molecule antagonists of HIV-1 latency. A more detailed analysis is provided for one of the hit compounds, antiviral 6 (AV6), which required nuclear factor of activated T cells for early mRNA expression while exhibiting RNA-stabilizing activity. It was found that AV6 reproducibly activated latent provirus from different lymphocyte-based clonal cell lines as well as from latently infected primary resting CD4(+) T cells without causing general T cell proliferation or activation. Moreover, AV6 complemented the latency antagonist activity of a previously described histone deacetylase (HDAC) inhibitor. This is a proof of concept showing that a high-throughput screen employing a cell-based model of HIV-1 latency can be utilized to identify new classes of compounds that can be used in concert with other persistent antagonists with the aim of viral clearance.

The discovery of a stable latent reservoir for HIV-1 in resting memory CD4(+) T cells provides a mechanism for lifelong persistence of HIV-1. The long-lived latently infected cells persist in spite of prolonged highly active antiretroviral therapy and present a major barrier to a cure of HIV-1 infection. In this review, we discuss the current understanding of HIV-1 persistence and latent viral infection in the context of effective antiretroviral therapy and the recent progress in purging latent viral reservoirs. Recent studies demonstrate that reactivation of latent HIV-1 is a promising strategy for the depletion of these viral reservoirs. A thorough evaluation of the anti-latency activity of drug candidates should include the measurement of changes in intracellular viral RNA, plasma virus levels, and the size of latent viral reservoirs, as well as potential adverse effects. Currently, there are several technical barriers to the evaluation of anti-latency drugs in vivo. We also discuss these challenging issues that remain unresolved.

Human immunodeficiency virus type 1 (HIV-1) establishes a latent reservoir in resting memory CD4(+) T cells. This latent reservoir is a major barrier to the eradication of HIV-1 in infected individuals and is not affected by highly active antiretroviral therapy (HAART). Reactivation of latent HIV-1 is a possible strategy for elimination of this reservoir. The mechanisms with which latency is maintained are unclear. In the analysis of the regulation of HIV-1 gene expression, it is important to consider the nature of HIV-1 integration sites. In this study, we analyzed the integration and transcription of latent HIV-1 in a primary CD4(+) T cell model of latency. The majority of integration sites in latently infected cells were in introns of transcription units. Serial analysis of gene expression (SAGE) demonstrated that more than 90% of those host genes harboring a latent integrated provirus were transcriptionally active, mostly at high levels. For latently infected cells, we observed a modest preference for integration in the same transcriptional orientation as the host gene (63.8% versus 36.2%). In contrast, this orientation preference was not observed in acutely infected or persistently infected cells. These results suggest that transcriptional interference may be one of the important factors in the establishment and maintenance of HIV-1 latency. Our findings suggest that disrupting the negative control of HIV-1 transcription by upstream host promoters could facilitate the reactivation of latent HIV-1 in some resting CD4(+) T cells.

The lysis of infected host cells by virus-specific cytolytic T lymphocytes (CTL) is an important factor in host resistance to viral infection. An optimal vaccine against human immunodeficiency virus type 1 (HIV-1) would elicit virus-specific CTL as well as neutralizing antibodies. The induction by a vaccine of HIV-1-specific CD8+ CTL in humans has not been previously reported. In this study, CTL responses were evaluated in HIV-1-seronegative human volunteers participating in a phase I acquired immune deficiency syndrome (AIDS) vaccine trial involving a novel vaccine regimen. Volunteers received an initial immunization with a live recombinant vaccinia virus vector carrying the HIV-1 env gene and a subsequent boost with purified env protein. An exceptionally strong env-specific CTL response was detected in one of two vaccine recipients, while modest but significant env-specific CTL activity was present in the second vaccinee. Cloning of the responding CTL gave both CD4+ and CD8+ env-specific CTL clones, permitting a detailed comparison of critical functional properties of these two types of CTL. In particular, the potential antiviral effects of these CTL were evaluated in an in vitro system involving HIV-1 infection of cultures of normal autologous CD4+ lymphoblasts. At extremely low effector-to-target ratios, vaccine-induced CD8+ CTL clones lysed productively infected cells present within these cultures. When tested for lytic activity against target cells expressing the HIV-1 env gene, CD8+ CTL were 3-10-fold more active on a per cell basis than CD4+ CTL. However, when tested against autologous CD4+ lymphoblasts acutely infected with HIV-1, CD4+ clones lysed a much higher fraction of the target cell population than did CD8+ CTL. CD4+ CTL were shown to recognize not only the infected cells within these acutely infected cultures but also noninfected CD4+ T cells that had passively taken up gp120 shed from infected cells and/or free virions. These results were confirmed in studies in which CD4+ lymphoblasts were exposed to recombinant gp120 and used as targets for gp120-specific CD4+ and CD8+ CTL clones. gp120-pulsed, noninfected targets were lysed in an antigen- specific fashion by CD4+ but not CD8+ CTL clones. Taken together, these observations demonstrate that in an in vitro HIV-1 infection, sufficient amounts of gp120 antigen are produced and shed by infected cells to enable uptake by cells that are not yet infected, resulting in the lysis of these noninfected cells by gp120-specific, CD4+ CTL.(ABSTRACT TRUNCATED AT 400 WORDS)

In recent decades, progress has been made worldwide in the prevention and control of HIV infection. What is the status of HIV prevention and control in China? This issue of HIV Medicine highlights the current progress in the management of and clinical research on HIV/AIDS in China, providing readers with a brief overview of this field. The Chinese authorities have made several efforts regarding the prevention and treatment of AIDS. For example, HIV infections transmitted by illegal blood transfusions have been eradicated, and HIV transmission among injecting drug users (IDUs) has been markedly reduced due to the implementation of a needle and syringe exchange programme. With the launch of the National Free Antiretroviral Treatment (ART) Program in 2003 and the subsequent implementation of the Four Free and One Care policy in 2006 [1], people living with HIV (PLWH) are often diagnosed via voluntary HIV testing or routine health examinations and provided free access to ART. The Four Free and One Care policy covers the provision of free ART to rural and urban residents without medical insurance; free voluntary counselling and HIV testing; free prevention of mother-to-child transmission; free schooling for orphaned children of PLWH; and care and economic assistance to PLWH with financial difficulties. In particular, the immediate initiation of ART in PLWH since 2016 has significantly reduced not only HIV transmission and prevalence but also AIDS-associated mortality [2]. Two studies in this issue analysed the clinical characteristics of patients receiving ART and pointed out the challenges faced by healthcare providers in China. Zhao et al. [3] presented the demographic and clinical characteristics of the national ART cohort for 2019. Their study showed great progress in increasing ART coverage and provided evidence of viral suppression across China. However, key populations of PLWH still face challenges: many are IDUs, have a history of treatment failure, or are co-infected with hepatitis C virus. Liu et al. [4] analysed the barriers to early diagnosis and timely ART initiation among PLWH, and their findings indicate the need for additional screening and intervention to improve the clinical management of HIV/AIDS. Co-infection with other viruses and bacteria among PLWH is associated with an increased incidence of failed immune reconstitution during ART and leads to a mortality rate higher than that associated with HIV infection alone. Two papers highlighted coinfections with hepatitis B virus (HBV) and Mycobacterium tuberculosis in PLWH. Jiang et al. [5] examined the factors associated with immune reconstitution in individuals with HIV/HBV co-infection receiving ART. They found that the baseline HIV viral load was the only significant factor that negatively influenced CD4 T-cell restoration in this population. Qi et al. [6] investigated tuberculosis (TB)-associated mortality within 90 days of admission and its risk factors among PLWH. Their data showed the 90-day mortality rate in patients with co-infection to be 13.6%, and early TB-associated death was associated with central nervous system TB, not receiving ART within 3 months after admission, and serum albumin levels < 25 g/L. In the era of ART, mother-to-child HIV transmission has substantially decreased. However, anti-HIV antibodies may still be detected in uninfected infants of mothers with chronic HIV infection. On monitoring the dynamics of anti-HIV antibodies in these infants, Liu et al. [7] noted the clearance of anti-HIV antibodies at more than 18 months post-delivery in 5.8% of uninfected children. These findings indicate that positive results for anti-HIV antibodies in infants aged 18–24 months should be carefully interpreted. Simultaneously, additional nucleic acid tests are necessary to confirm a diagnosis of HIV infection. Although ART has increased the life expectancy and improved the quality of life of PLWH, non-AIDS-associated events remain an issue. Lin et al. [8] summarized the clinical characteristics of non-AIDS-associated events involving multiple organs, such as the liver and kidneys, and suggested that multidisciplinary management is necessary for PLWH. Ying et al. [9] investigated the epidemiology, clinical features, and prognostic factors of HIV-associated talaromycosis and found an increased prevalence in Guangdong, China. Typical skin lesions were noted in 44.5% of patients. Induction therapy with azole alone was associated with a higher mortality than therapy with amphotericin B deoxycholate. These results may serve as a guide for clinicians in the management of HIV-associated talaromycosis. The viral reservoir is the biggest hurdle in the race to cure HIV/AIDS. In the shock and kill strategy, the latent HIV reservoir is maximally activated by stimulation with latency reversal agents and further cleared by host immune surveillance. Li et al. [10] evaluated the safety and efficacy of chidamide, a histone deacetylase inhibitor, for HIV-1 latency reversal in seven participants with viral suppression. All participants exhibited robust and repeated plasma viral rebound and increased cell-associated HIV-1 RNA levels, but only grade 1 adverse events were reported. Chidamide seems to be an effective agent in disrupting HIV-1 latency and reducing HIV-1 DNA loads; however, these findings need to be confirmed in randomized controlled clinical trials. Zhang et al. [11] summarized recent findings regarding the role of CD8 T-cells in controlling HIV infection, highlighting the differences between conventional antigen-specific and innate-like CD8 T-cells. Antiviral activity of CD8 T-cells in PLWH receiving ART may not be achieved via an antigen-specific approach, as HIV-specific CD8 T-cells can sense, but not effectively eliminate, cells harbouring intact proviruses. By contrast, virtual memory CD8 T-cells, a semi-differentiated subset of CD8 T-cells, may be involved in controlling the HIV DNA reservoir in patients receiving ART. In China, although remarkable progress has been made in controlling the HIV epidemic, challenges remain. First, the absolute number of PLWH is approximately 1.2 million, and the annual number of newly identified HIV/AIDS cases has been determined to be 150 000 in the last 3 years. In particular, approximately 31% of PLWH were diagnosed in the advanced stage, with peripheral CD4 T-cell counts of less than 200 cells/µL [3, 12]. In-time HIV screening is necessary to identify PLWH at an early stage. Second, the choice of ART drugs for PLWH is limited; for example, integrase inhibitors have recently been included in the free ART regimen, but this programme does not cover all PLWH. Third, sexual transmission has become the main route of HIV spread, and such transmission needs to be reduced by efficient preventive programmes, such as those providing universal pre-exposure prophylaxis. The 90-90-90 goals put forth by the Joint United Nations Program on AIDS/HIV seek for 90% of PLWH to be diagnosed, 90% of those diagnosed to receive ART, and 90% of those receiving ART to show viral suppression. It is feasible to achieve these 90-90-90 goals during next 5 years in China, but we still have a long way to go for the development of a cure for HIV infection.

Lentiviruses are retroviruses that primarily infect myeloid cells, leading to acute inflammatory infections in many tissues particularly, lung, joints and the central nervous system (CNS). Acute infection by lentiviruses is followed by persistent/latent infections that are not cleared by the host immune system. HIV and SIV are lentiviruses that also infect CD4+ lymphocytes as well as myeloid cells in blood and multiple tissues. HIV infection of myeloid cells in brain, lung and heart cause tissue specific diseases as well as infect cells in gut, lymph nodes and spleen. AIDS dementia and other tissue specific disease are observed when infected individuals are immunosuppressed and the number of circulating CD4+ T cells declines to low levels. Antiretroviral therapy (ART) controls viral spread and dramatically changes the course of immunodeficiency and AIDS dementia. However, ART does not eliminate virus-infected cells. Brain macrophages contain HIV DNA and may represent a latent reservoir that persists. HIV latency in CD4+ lymphocytes is the main focus of current research and concern in efforts to eradicate HIV. However, a number of studies have demonstrated that myeloid cells in blood and tissues of ART suppressed individuals harbor HIV DNA. The resident macrophages in tissues such as brain (microglia), spleen (red pulp macrophages) and alveolar macrophages in lung are derived from the yolk sac and can self renew. The question of the latent myeloid reservoir in HIV has not been rigorously examined and its potential as a barrier to eradication been considered. Using a well characterized SIV ART suppressed, non-human primate (NHP) model, our laboratory developed the first quantitative viral outgrowth assay (QVOA) designed to evaluate latently infected CD4+ lymphocytes and more recently developed a similar protocol for the assessment of latently infected myeloid cells in blood and brain. Using an SIV ART model, it was demonstrated that myeloid cells in blood and brain harbor latent SIV that can be reactivated and produce infectious virus in vitro. These studies demonstrate for the first time that myeloid cells have the potential to be a latent reservoir of HIV that produces infectious virus that can be reactivated in the absence of ART and during HIV eradication strategies. Graphical Abstract.

Viral reservoirs, residual viremia, and the potential of highly active antiretroviral therapy to eradicate HIV infection