Carolina Garrido

Eradicating HIV-1 infection: seeking to clear a persistent pathogen

Effective antiretroviral therapy (ART) blunts viraemia, which enables HIV-1-infected individuals to control infection and live long, productive lives. However, HIV-1 infection remains incurable owing to the persistence of a viral reservoir that harbours integrated provirus within host cellular DNA. This latent infection is unaffected by ART and hidden from the immune system. Recent studies have focused on the development of therapies to disrupt latency. These efforts unmasked residual viral genomes and highlighted the need to enable the clearance of latently infected cells, perhaps via old and new strategies that improve the HIV-1-specific immune response. In this Review, we explore new approaches to eradicate established HIV-1 infection and avoid the burden of lifelong ART.

Dual-Affinity Re-Targeting proteins direct T cell–mediated cytolysis of latently HIV-infected cells

Enhancement of HIV-specific immunity is likely required to eliminate latent HIV infection. Here, we have developed an immunotherapeutic modality aimed to improve T cell-mediated clearance of HIV-1-infected cells. Specifically, we employed Dual-Affinity Re-Targeting (DART) proteins, which are bispecific, antibody-based molecules that can bind 2 distinct cell-surface molecules simultaneously. We designed DARTs with a monovalent HIV-1 envelope-binding (Env-binding) arm that was derived from broadly binding, antibody-dependent cellular cytotoxicity-mediating antibodies known to bind to HIV-infected target cells coupled to a monovalent CD3 binding arm designed to engage cytolytic effector T cells (referred to as HIVxCD3 DARTs). Thus, these DARTs redirected polyclonal T cells to specifically engage with and kill Env-expressing cells, including CD4+ T cells infected with different HIV-1 subtypes, thereby obviating the requirement for HIV-specific immunity. Using lymphocytes from patients on suppressive antiretroviral therapy (ART), we demonstrated that DARTs mediate CD8+ T cell clearance of CD4+ T cells that are superinfected with the HIV-1 strain JR-CSF or infected with autologous reservoir viruses isolated from HIV-infected-patient resting CD4+ T cells. Moreover, DARTs mediated CD8+ T cell clearance of HIV from resting CD4+ T cell cultures following induction of latent virus expression. Combined with HIV latency reversing agents, HIVxCD3 DARTs have the potential to be effective immunotherapeutic agents to clear latent HIV-1 reservoirs in HIV-infected individuals.

Expanded Cytotoxic T-cell Lymphocytes Target the Latent HIV Reservoir

Enhanced human immunodeficiency virus (HIV)-specific immunity may be required for HIV eradication. Administration of autologous, ex vivo expanded, virus-specific, cytotoxic T-lymphocytes derived from HIV-infected patients on suppressive antiretroviral therapy (HXTCs) are a powerful tool for proof-of-concept studies. Broadly specific, polyclonal HXTCs resulting from ex vivo expansion demonstrated improved control of autologous reservoir virus compared to bulk CD8(+) T cells in viral inhibition assays. Furthermore, patient-derived HXTCs were able to clear latently infected autologous resting CD4(+) T cells following exposure to the latency-reversing agent, vorinostat. HXTCs will be ideal reagents to administer with precise control in future in vivo studies in combination with latency-reversing agents.

Peripheral Vγ9Vδ2 T Cells Are a Novel Reservoir of Latent HIV Infection.

Eradication of HIV infection will require the identification of all cellular reservoirs that harbor latent infection. Despite low or lack of CD4 receptor expression on Vδ2 T cells, infection of these cells has previously been reported. We found that upregulation of the CD4 receptor may render primary Vδ2 cells target for HIV infection in vitro and we propose that HIV-induced immune activation may allow infection of γδ T cells in vivo. We assessed the presence of latent HIV infection by measurements of DNA and outgrowth assays within Vδ2 cells in 18 aviremic patients on long-standing antiretroviral therapy. In 14 patients we recovered latent but replication-competent HIV from highly purified Vδ2 cells demonstrating that peripheral Vδ2 T cells are a previously unrecognized reservoir in which latent HIV infection is unexpectedly frequent.

Broadly-specific Cytotoxic T Cells Targeting Multiple HIV Antigens Are Expanded From HIV+ Patients: Implications for Immunotherapy

Antiretroviral therapy (ART) is unable to eradicate human immunodeficiency virus type 1 (HIV-1) infection. Therefore, there is an urgent need to develop novel therapies for this disease to augment anti-HIV immunity. T cell therapy is appealing in this regard as T cells have the ability to proliferate, migrate, and their antigen specificity reduces the possibility of off-target effects. However, past human studies in HIV-1 infection that administered T cells with limited specificity failed to provide ART-independent, long-term viral control. In this study, we sought to expand functional, broadly-specific cytotoxic T cells (HXTCs) from HIV-infected patients on suppressive ART as a first step toward developing cellular therapies for implementation in future HIV eradication protocols. Blood samples from seven HIV+ patients on suppressive ART were used to derive HXTCs. Multiantigen specificity was achieved by coculturing T cells with antigen-presenting cells pulsed with peptides representing Gag, Pol, and Nef. All but two lines were multispecific for all three antigens. HXTCs demonstrated efficacy as shown by release of proinflammatory cytokines, specific lysis of antigen-pulsed targets, and the ability to suppress HIV replication in vitro. In conclusion, we are able to generate broadly-specific cytotoxic T cell lines that simultaneously target multiple HIV antigens and show robust antiviral function.

HIV latency-reversing agents have diverse effects on natural killer cell function

In an effort to clear persistent HIV infection and achieve a durable therapy-free remission of HIV disease, extensive pre-clinical studies and early pilot clinical trials are underway to develop and test agents that can reverse latent HIV infection and present viral antigen to the immune system for clearance. It is, therefore, critical to understand the impact of latency-reversing agents (LRAs) on the function of immune effectors needed to clear infected cells. We assessed the impact of LRAs on the function of natural killer (NK) cells, the main effector cells of the innate immune system. We studied the effects of three histone deacetylase inhibitors [SAHA or vorinostat (VOR), romidepsin, and panobinostat (PNB)] and two protein kinase C agonists [prostratin (PROST) and ingenol] on the antiviral activity, cytotoxicity, cytokine secretion, phenotype, and viability of primary NK cells. We found that ex vivo exposure to VOR had minimal impact on all parameters assessed, while PNB caused a decrease in NK cell viability, antiviral activity, and cytotoxicity. PROST caused non-specific NK cell activation and, interestingly, improved antiviral activity. Overall, we found that LRAs can alter the function and fate of NK cells, and these effects must be carefully considered as strategies are developed to clear persistent HIV infection.

Gold nanoparticles to improve HIV drug delivery

BACKGROUND Antiretroviral therapy (ART) has improved lifespan and quality of life of patients infected with the HIV-1. However, ART has several potential limitations, including the development of drug resistance and suboptimal penetration to selected anatomic compartments. Improving the delivery of antiretroviral molecules could overcome several of the limitations of current ART. RESULTS & CONCLUSION Two to ten nanometer diameter inorganic gold crystals serve as a base scaffold to combine molecules with an array of properties in its surface. We show entry into different cell types, antiviral activity of an HIV integrase inhibitor conjugated in a gold nanoparticle and penetration into the brain in vivo without toxicity. Herein, gold nanoparticles prove to be a promising tool to use in HIV therapy.

Translational challenges in targeting latent HIV infection and the CNS reservoir problem.

Too controversial to discuss only a short time ago, achieving a cure for HIV infection has become a priority in HIV research. However, substantial challenges must be overcome. Among key hurdles to be surmounted is the definition of a reliable, validated model in which to test latency reversal agents (LRAs), as current primary cell models differ in their response to such agents. Animal models such as the HIV-infected humanized BLT mouse and SIV-infected macaque will be essential to study LRAs and to quantify their effects in anatomic reservoirs. Of several potential anatomic reservoirs, the central nervous system presents a significant obstacle, as it is known to harbor persistent HIV infection and is difficult to access for study and therapeutic intervention.

Detection of human immunodeficiency virus RNAs in living cells using Spinach RNA aptamers

Many techniques currently used to measure HIV RNA production in cells suffer from limitations that include high background signal or the potential to destroy cellular context. Fluorophore-binding RNA aptamers offer the potential for visualizing RNAs directly in living cells with minimal cellular perturbation. We inserted a sequence encoding a fluorophore-binding RNA aptamer, known as Spinach, into the HIV genome such that predicted RNA secondary structures in both Spinach and HIV were preserved. Chimeric HIV-Spinach RNAs were functionally validated in vitro by testing their ability to enhance the fluorescence of a conditional fluorophore (DFHBI), which specifically binds Spinach. Fluorescence microscopy and PCR were used to verify expression of HIV-Spinach RNAs in human cells. HIV-1 gag RNA production and fluorescence were measured by qPCR and fluorometry, respectively. HIV-Spinach RNAs were fluorometrically detectable in vitro and were transcribed in human cell lines and primary cells, with both spliced and unspliced species detected by PCR. HIV-Spinach RNAs were visible by fluorescence microscopy in living cells, although signal was reproducibly weak. Cells expressing HIV-Spinach RNAs were capable of producing fluorometrically detectable virions, although detection of single viral particles was not possible. In summary, we have investigated a novel method for detecting HIV RNAs in living cells using the Spinach RNA aptamer. Despite the limitations of the present aptamer/fluorophore combination, this is the first application of this technology to an infectious disease and provides a foundation for future research into improved methods for studying HIV expression.

γδ T cells: an immunotherapeutic approach for HIV cure strategies

Current strategies aimed to cure HIV infection are based on combined efforts to reactivate the virus from latency and improve immune effector cell function to clear infected cells. These strategies are primarily focused on CD8+ T cells and approaches are challenging due to insufficient HIV antigen production from infected cells and poor HIV-specific CD8+ T cells. γδ T cells represent a unique subset of effector T cells that can traffic to tissues, and selectively target cancer or virally infected cells without requiring MHC presentation. We analyzed whether γδ T cells represent a complementary/alternative immunotherapeutic approach towards HIV cure strategies. γδ T cells from HIV-infected virologically suppressed donors were expanded with bisphosphonate pamidronate (PAM) and cells were used in autologous cellular systems ex vivo. These cells (a) are potent cytotoxic effectors able to efficiently inhibit HIV replication ex vivo, (b) degranulate in the presence of autologous infected CD4+ T cells, and (c) specifically clear latently infected cells after latency reversal with vorinostat. This is the first proof of concept to our knowledge showing that γδ T cells target and clear autologous HIV reservoirs upon latency reversal. Our results open potentially new insights into the immunotherapeutic use of γδ T cells for current interventions in HIV eradication strategies.