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Basic Biomedical Sciences Research

These pilot awards support innovative early-stage laboratory exploration aimed at understanding mechanisms of HIV prevention, treatment, or cure at the cellular or subcellular level. The proposals address a significant question or barrier in HIV science, and is highly likely to yield findings that will serve as a basis for compelling studies of larger magnitude. The lay abstracts below were provided by the principal investigators at the time of application.


In 2024, applications for these biannual awards were newly restricted to early career stage investigators (ESI) with named mentors.

Enhancing Anti-HIV Gene Therapy Through Epigenetic Modulation

Principal Investigator:  Mohamed Bouzidi, PhD, Vitalant Research Institute

Budget:  $270,000

Start Date: February 1, 2024        End Date: January 31, 2026

Project Abstract:  CRISPR-Cas9-mediated excision of the HIV provirus is a promising cure strategy. However, little is known about the actual efficacy of this approach in people living with HIV (PLWH). Reports have shown that HIV latency, the main barrier to an HIV cure, is maintained through chromatin condensation at the integration site and methylation of the provirus, making it inaccessible to CRISPR-Cas9 complexes. Taken together, it appears unlikely that the CRISPR-Cas9 approach alone will be sufficient to achieve a cure for HIV infection. Several methylation and chromatin state modulators, such as romidepsin and decitabine, are used in cancer treatment and have been proposed as latency reversal agents (LRA). Building on our promising previous work and preliminary data, we propose to investigate how pharmacological manipulation of DNA methylation and chromatin state can improve anti-HIV gene therapy. Under the mentorship of Dr. Satish Pillai, I will use DNA methyltransferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors to improve CRISPR-Cas9 editing of the HIV provirus. We will administer DNMT inhibitors, inducing genome-wide hypomethylation, to primary CD4+ T cells and nucleofect them with anti-HIV Cas9 ribonucleoproteins (Cas9-RNPs). We will apply targeted bisulfite sequencing and Tracking of Indels by Decomposition (TIDE) analysis to evaluate the impact of methylation on gene editing. We will transduce J-Lat cells (latently-infected Jurkat cells) with the CRISPRoff-V2.1/TeTv4 system, consisting of methylation or demethylation machinery, respectively fused with a dead Cas9, to hyper- and hypomethylate specific sites in the provirus and apply the previously described approach. Nucleofection of a panel of J-Lat clones, characterized by unique integration sites, with anti-HIV Cas9-RNPs followed by assay for transposase accessibility and sequencing (ATAC-seq) and Tracking of Indels by DEcomposition (TIDE) analysis will be used to determine the impact of chromatin state on anti-HIV gene editing. Additionally, we will administer HDAC inhibitors to latently infected primary CD4+ T cells and use the same method to confirm our observations ex vivo. We expect to observe higher editing efficiency in proviruses harboring unmethylated CpG islands, located in open chromatin regions. Ultimately, this project may lead to key enhancements in anti-HIV gene therapy.

Enhancing Anti-HIV Immunity through Multilineage CAR Cells Derived from Hematopoietic Stem Cells

Principal Investigator:  Wenli Mu, PhD, UC Los Angeles

Budget:  $269,998

Start Date: February 1, 2024        End Date: January 31, 2026

Project Abstract:  Hematopoietic stem and progenitor cell (HSPC) transplantation underlies a HIV cure reported in at least 3 individuals to date. HSPC-based gene therapy can support lifelong generation of functional immune progeny. Whereas peripheral T cell gene therapy often involves ex vivo non-physiologic stimulation that skews T cell phenotypes and alters function and persistence in vivo, HSPCs display lifelong self-renewal properties and produces naturally developed, functionally normal progeny. Importantly, CD4-chimeric antigen receptor (CAR)-modified HSPCs can differentiate into multiple hematopoietic lineages, including T cells, macrophages and NK cells in humanized BLT mice and nonhuman primates (NHPs), suggesting that CAR-engineered HSPCs are capable of providing greater and broader immune responses to combat HIV. Our preliminary data demonstrates that CD4-based CAR macrophages (MQs) can direct phagocytosis and cytotoxicity against HIV envelope-expressing cells in vitro. However, besides CAR-expressing T cells, HSPC-derived CAR cells in other lineages have not been fully characterized, and their functionality as well as their interaction with CAR T cells remains unknown. Our hypothesis is that CAR-expressing natural killer (NK) cells and MQs constantly produced from gene modified HSPCs can provide innate immune surveillance benefits by facilitating antigen presentation and secreting pro-inflammatory cytokine to boost both CAR T and endogenous CTL function while targeting different anatomical reservoirs. We propose by carefully selecting CAR molecule candidate domains that can enhance whole-cell phagocytosis and effector functions, we aim to optimize the function of anti-HIV CAR-MQs and CAR-NK to ultimately improve their therapeutic potential and provide critical antiviral activities in synergy with CAR-T cells. By transducing HSPCs with optimized CD4-based CAR constructs, we aim to provide a lifelong source of CAR effector cells in multiple lineages capable of suppressing/eliminating HIV-1 replication to achieve functional cure.


Probing the HIV Viral Reservoir for HIV-Specific Killer Cells

Principal Investigator:  Jennifer Dan, MD PhD, UC San Diego

Budget:  $270,000

Start Date: February 1, 2024        End Date: January 31, 2026

Project Abstract:  One of the foremost barriers to HIV cure is the latent viral reservoir. Despite antiretroviral therapy (ART), HIV lies dormant within tissues and various immune cells. Once ART is stopped, HIV re-emerges from these tissues and immune cells. Specifically, HIV lies latent within T follicular helper (Tfh) cells. A Tfh cell is a specialized CD4+ T cell whose main function is to instruct nearby B cells within germinal centers (GC). This results in B cell differentiation into memory B cells and plasma cells and the development of high affinity antibodies, critical processes of acquired immunity. However, HIV-infected GC Tfh cells have diminished Tfh function compared to uninfected Tfh cells, permitting persistence. We have developed the Activation Induced Marker (AIM) assay to identify antigen-specific CD4+ T cells and GC Tfh cells in secondary lymphoid tissue. In addition to the traditional Tfh cells and regulatory Tfh cell, we were the first to describe the phenomenon of a “killer” Tfh cell. These “killer” Tfh cells are similar to cytolytic CD4+ T cells in circulation. HIV-specific cytolytic CD4+ T cells are capable of killing HIV-infected CD4+T cells and controlling infection. Higher frequencies of HIV-specific cytolytic CD4+ T cells have been observed in long term non-progressors and elite controllers. We hypothesize that “killer” Tfh cells within lymphoid tissues are akin to cytolytic CD4+T cells in circulation with the ability to obliterate the latent viral reservoir. The NIH-funded Last Gift Study is ongoing in San Diego and provides a unique opportunity to understand the HIV reservoir across the human body. Last Gift participants, upon their death, undergo a rapid research autopsy within 6 hours, which allows collection of fresh tissue with optimal cellular viability and protein and nuclei acids integrity. The rapid autopsy team collects tissues extensively throughout the entire body including entire lymph nodes, which can be used for this pilot project to probe the latent HIV reservoir. Under the mentorship of Dr. Davey Smith, we will assess HIV-specific Tfh cells in fresh lymphoid tissues collected from 10 Last Gift participants. Our long-term goal is to show that HIV-specific “killer Tfh” cells exist within HIV lymph nodes and can be harnessed to kill HIV-infected GC Tfh cells, thereby providing a means to eliminate the latent HIV reservoir.


Spinal Cord as a Distinct Site of HIV Persistence and Dispersal

Principal Investigator:  Mattia Trunfio, DTMH MD(c), UC San Diego

Budget:  $270,000

Start Date: February 1, 2024        End Date: January 31, 2026

Project Abstract:  Despite the abundance of data about HIV infection in the central nervous system (CNS), most studies focus on the brain and neglect the spinal cord. Investigations on neurocognitive complications, HIV persistence, or antiretrovirals (ARVs) penetration into the CNS have mostly used cerebrospinal fluid (CSF) and brain tissues. Our preliminary data reported higher HIV DNA levels in the spinal cord compared to brain tissues with heterogeneity in HIV reservoirs across brain areas. Compared to the brain, the spinal cord has its own peculiarities that encompass the blood-spinal cord barrier and the tissue-specific abundance and functioning of macrophages and microglia, which carry the highest burden of HIV in the CNS. Therefore, neither ARVs penetration nor the proviral dynamics in the spinal cord can be inferred from what is known for other CNS parts. Our overarching hypothesis is that the spinal cord is a distinct site of HIV persistence compared to other CNS regions (in terms of size, activity, and diversity), that it represents an independent source of viral dispersal when ARVs are interrupted, and that measures of HIV persistence and dispersal are influenced by ARVs tissue levels and inflammation. To investigate these hypotheses, we will analyze flash frozen tissues from the pons, medulla, and the cervical, thoracic, and lumbar segments of the spinal cord of 15 people with HIV (PWH) enrolled in the Last Gift program. The Last Gift program (San Diego) enrolls altruistic PWH with a life-shortening illness who participate in HIV cure research at the end-of-life, including full body donation for a rapid research autopsy. As part of this proposed project, we will 1) measure levels of HIV RNA and HIV DNA (ddPCR), and proviral diversity (full length HIV DNA single genome sequencing) in various spinal segments, 2) evaluate how these reservoir measures relate to CSF inflammation and ARVs tissue levels, 3) determine if the spinal cord is a source of HIV re-seeding in PWH who voluntarily interrupt ARVs before death. We will compare these data with similar reservoir data generated from brain, lymph tissues, blood, and CSF made available as part of the ongoing NIAID-funded program (AI169609). Our results will inform larger studies addressing HIV reservoirs in the CNS with the aim of developing strategies to effectively clear HIV infection. Mentor: Prof. Antoine Chaillon.


Principal Investigator:  Yusuke Matsui, PhD, J. David Gladstone Institutes

Budget:  $270,000

Start Date: February 1, 2024        End Date: January 31, 2026

Project Abstract:  The brain is an understudied target organ for HIV-1, and ~50% of patients on antiretroviral therapy experience neurocognitive disorders, known as HIV-associated neurocognitive disorder (HAND). Microglia are brain-resident macrophages and the target cells for HIV-1. Microglia are heterogeneous; they interact with surrounding neurons and astrocytes and alter their gene expression profiles in response to environmental cues, forming characteristic “subsets” based on transcriptome analysis. Here, we hypothesize that subset characteristics of microglia influence susceptibility to HIV-1 and the establishment and maintenance of latent infection. Determining the precise identity of the microglia reservoir is critical for our understanding of how latency is established and maintained in the brain and for the development of effective treatment aimed at HIV-1 eradication in central nervous system (CNS). We propose to test our hypothesis using brain organoids containing microglia. Brain organoids containing microglia are being generated from human induced pluripotent stem (iPS) cells and are emerging as a new tool to study brain disorders in HIV infection. Presently, microglial subsets have not been mapped in brain organoids, but transcriptome and cell surface marker analyses suggest that brain organoids reproduce the physiological brain environment. Also, iPSC-derived macrophages contained in alveolar organoids successfully reproduce in vivo subset characterization. We will generate two types of brain organoids containing microglia and classify subsets of microglia based on transcriptome expression. We will combine this technology with cutting-edge single-cell RNA sequencing, CRISPR-Cas9 knockout experiments, light-sheet microscopy, and infections with dual-fluorescent HIV clones. Our studies are significant because they address an important remaining hurdle in curing HIV: characterizing and eradicating the viral reservoir in the brain. They are innovative because they challenge the status quo by defining subset characteristics in brain organoids with microglia. After completion of our studies, we will have accomplished mechanistic understanding that will lead to new HIV-1 eradication strategies against viral reservoir of CNS by characterizing subsets of microglia that are the breeding ground for latent infection in the brain. Proposed mentor's name: Melanie Ott


Chimeric Antigen Receptors on NK Cells to Combat HIV Infection

Principal Investigator:  Jocelyn Kim, MD PhD, UC Los Angeles

Budget:  $270,000

Start Date: February 1, 2024        End Date: January 31, 2026

Project Abstract:  In 2020, 38 million people worldwide were living with HIV/AIDS. Antiretroviral therapy (ART) can suppress but not cure HIV infection, mainly because of the persistence of infected cells. These cells arise from the ability of HIV to integrate its viral genome into the host cell’s chromosomes and remain quiescent and elude the immune system. This reservoir of latently infected cells is the most formidable obstacle to curing HIV. One approach to eliminating latently infected cells is a “kick and kill” strategy, in which latent cells are induced to produce viral proteins by a latency reversing agent (LRA).  Our collaborator Dr. Paul Wender, an expert synthetic chemist, synthesized the first bryostatin-1 analog (SUW133) as a superior LRA compared to its parent compound. However, SUW133 reversed latent cells in vivo, but not all infected cells emerging from latency were killed.  To this end, we investigated whether natural killer (NK) cells could help kill these persistent infected cells. NK cells are immune cells that inherently control HIV infection. Due to their intrinsic anti-viral and anti-cancer function, allogeneic NK cells are already under clinical investigation to treat COVID-19 patients and have been safely and effectively used in patients with leukemia.  However, chronic HIV infection results in their loss of function, which is not completely restored by suppression of viral replication by ART.  Thus, we suspected allogeneic NK cells from healthy human donors may be an attractive source for providing a potent anti-HIV killing agent. Interestingly, we found a kick and kill strategy using the LRA SUW133 as the “kick” and human allogeneic NK cells as the “kill” yielded remarkable results compared to the LRA or NK cells alone with substantial delays in viral rebound after stopping ART in HIV-infected humanized mouse series.  Importantly, this kick and kill strategy cleared HIV infection from 40% of infected animals. This work thus far provides proof-of-concept that NK cells plus an LRA leads to unprecedented in vivo reservoir reduction, a task nearly insurmountable in the field of HIV eradication. Further research to improve this strategy is urgently needed. Here our overall objective is to engineer and improve NK cells to efficiently fight HIV infection.


Characterization of Provirus-Host Chromatin Interaction to Achieve an HIV Cure

Principal Investigator:  Sarah LaMere, PhD DVM, UC San Diego

Budget:  $270,000

Start Date: February 1, 2024        End Date: January 31, 2026

Project Abstract:  Despite decades of research into the mechanisms of HIV persistence, a cure remains elusive. Basic biomedical research has focused strongly on the mechanisms of viral infection and replication, leading to the development of many successful antiretroviral therapies and allowing people with HIV to live without fear of developing AIDS as long as they maintain their medication regimen. However, there are two large obstacles that have prevented the advancement of cure research: 1) poor understanding of the epigenetic regulation of the provirus and the interacting host chromatin, which impacts our ability to manipulate, excise, or silence the provirus and 2) poor characterization of the latent reservoir throughout the body due to limited access to rapid autopsy specimens, which largely impacts the success of drug delivery. Under the guidance of Dr. Davey Smith, I propose to address both of these hurdles by making use of recent advancements in single cell techniques and high-resolution chromatin characterization to investigate putative epigenetic mechanisms in tissues. These tissue reservoirs are critical, as they likely contribute to repopulation of systemic HIV upon discontinuation of ART. This project will take advantage of samples collected from the ‘Last Gift’ cohort co-created by Dr. Smith, which entails that participants provide their entire bodies after they die for a rapid research autopsy protocol. Data generated from this proposal will be integrated with data generated as part of the parent grant, which already includes cell-associated viral RNA expression, proviral load, T cell receptor sequencing, integration site sequencing, and full-length HIV genome sequencing. We hypothesize that the integrated HIV provirus impacts host chromatin conformation, requiring a 3D examination extending beyond simple epigenetic modifications. Further, we hypothesize that this impact depends heavily upon both integration site locale and cell type. Therefore, we propose to examine the impact of the provirus upon 3D chromatin conformation and associated epigenetic modifications alongside integration sites in both a CD4 T cell latency model and then in two separate tissues documented to have evidence of clonal expansion from two Last Gift participants.



Basic Biomedical Discovery Initiative 2022

Single-Cell HIV Reservoir Dynamics Following Gene Modified Autologous Stem Cell Transplantation
Timothy Henrich, MD MMDc
UC San Francisco

The use of gene-modified cells to reduce HIV reservoir size and delay HIV recrudescence is on the forefront of HIV curative science. However, current approaches may have limited impact in people with HIV (PWH) and may only provide a single layer of protection against infection.  As a result, the AMC #097 study, “A Phase I Study of Stem Cell Gene Therapy for HIV Mediated by Lentivector Transduced, Pre-Selected CD34+ Cells: A Trial of the AIDS Malignancy Consortium,” was implemented to use multiple anti-HIV gene-modified cells to block HIV infection at different states of the viral life cycle. We hypothesize that the combined gene-modification approach in the AMC097 study will lead to reductions in the overall burden of HIV infection and lead to post-treatment control during analytical treatment interruption (ATI). Furthermore, we have developed a novel assay to determine if HIV infects gene-modified cells. Our aims are to determine the long-term impact of autologous SCT with gene-modified stem cells simultaneously targeting different stages of the HIV replication cycle on the burden of HIV infection, and to determine if gene-modified cells become infected prior to and following cessation of ART in the AMC097 trial. 


Neuroimaging and Inflammation among People Living with HIV with History of Methamphetamine Use
Judith Lobo, PhD
UC San Diego

Methamphetamine (METH) is the most commonly used illicit substance in San Diego and there is limited treatment available for METH dependence. Understanding the how METH use changes the way the brain works is an essential step in developing the best patient care possible. Based on previous studies using brain imaging, we hypothesize that the same brain regions are negatively affected by HIV status, history of METH-use, and inflammation. However, little is known about how the brain behaves when an individual is both living with HIV and has a history of METH use. Our goal is to help better understand how brain activity is changed in this setting, and whether those changes are due to HIV status, METH use or both. Inflammation is thought to be related to these brain function alterations, so we will use inflammatory levels measured by blood sample to determine if these changes are related to increased levels of inflammation. The data used in this study has already been collected, by the University of California San Diego: Translational Methamphetamine AIDS Research Center. We will add to this dataset by running more inflammatory marker-panels on frozen blood samples. In order to be able to compare the effects of HIV and METH, we will compare the data of individuals in four groups: 1) HIV+ METH+, 2)HIV- METH+, 3) HIV+ METH-, 4) HIV- METH-. Our neuroimaging analysis will focus on two brain regions, the dorsolateral prefrontal cortex and the anterior cingulate cortex. To our knowledge, this would be the first neuroimaging and inflammation study in this population. The goal of this work is to help better understand the complex relationships between substance use, inflammation, and the brain in people with HIV who also have a history of METH use, a growing population in California.


Cannabinoid Receptor 2-Signaling in Endothelial Integrity and Functions in the Context of HIV
Maria Cecilia Marcondes, PhD
Ballad Research Institute, San Diego Biomedical Research Institute

Cannabis is broadly used by persons living with HIV. The mechanisms by which cannabinoids harm or benefit the brain in the context of HIV have yet to be defined, particularly on endothelial function and the blood brain barrier (BBB). Our goal is to examine the influence of cannabis, via CB2R-signaling, on vascular integrity and function, in the context of HIV, with implications on cell migration and penetration of antiretroviral drugs (ART). The project is novel for multiple reasons: (i) it will address a knowledge gap on the effects of cannabinoids on BBB molecular and functional properties in the context of HIV, (ii), it will build on evidence of a beneficial role for CB2R stimulation in the context of HIV infection, to recover damaged endothelium, (iii)it will utilize gene editing strategies to produce cell line tools to study outcomes influenced by CBR2, (iv) it will provide mechanistic and translational information on the impact of cannabis on eradication strategies such as ART penetration through the BBB to target latent CNS reservoirs, (v) it will employ clinically-relevant systems, with a multidisciplinary team, and state of the art technical expertise, and (v)it will test biomarkers of intact BBB in the context of HIV and cannabis.


Modulating Therapeutic HIV Vaccine Responses Using Rapamycin to Achieve Post-Treatment Control
Gema Mendez Lagares, PhD
UC Davis

Most HIV-infected patients who stop taking antiretroviral drugs experience rapid viral rebound and suffer in the longer term from presence of more virus in blood and lower CD4+ T-cell counts. HIV-1 infection generally increases T-cell metabolism, which means that cells are more active, which promotes successful viral integration and replication. Our preliminary data show that although therapeutic T-cell vaccination can effectively generate cytotoxic “killer” T-cells that kill HIV-infected cells, in most cases this antiviral activity is insufficient to limit viral replication. It is critical to improve upon these promising vaccine strategies and to design novel approaches for enhancing vaccine-induced immunity. Given intriguing data indicating that rapamycin, a drug that targets T-cell metabolism and is used at high doses as an immunosuppressive agent, can enhance the magnitude and the quality of vaccine-induced virus specific memory T-cells, we hypothesized that stringent post-treatment control requires both effective anti-SIV responses to a therapeutic vaccine and unfavorable metabolic conditions for viral replication. Here we propose to modulate the mTOR pathway during antiretroviral therapy interruption, to reduce T-cell activation and the availability of new target cells, and during vaccination, to enhance SIV-specific cellular immunity. To achieve our goal, we will investigate two important effects of rapamycin in rhesus macaques; 1) its effect in controlling inflammation and immune activation and 2) its ability to create effective T-cell responses in recipients of therapeutic vaccination.


A Novel Primary Cell HIV-CRISPR Screen to Elucidate Mechanisms of a Noncytotoxic Antiviral Response
Ujjwal Rathore, PhD
David Gladstone Institutes

HIV attacks the body’s immune system by infecting a specific type of immune cells called CD4 T-cells. If left untreated, the destruction of CD4 T-cells makes it difficult for the body to fight infections, a condition known as Acquired Immunodeficiency Syndrome (AIDS). Despite 40 years of research, we still do not have a cure for HIV. Current anti-HIV drugs (also known as antiretrovirals) keep the HIV virus under control but cannot eliminate it from the body, necessitating a life-long dependence on these drugs. However, some rare HIV-positive individuals can stay symptom-free and disease-free for years without any antiretroviral therapy and are called elite controllers or HIV long-term non-progressors. If we can crack the mystery of how these individuals keep HIV under control without drugs, then we can use that knowledge to find a cure for HIV. In some of these HIV controllers, it was found that their CD8 T-cells, another type of immune cells important for killing virus-infected cells in the body, can stop the HIV virus from multiplying in the HIV-infected CD4 cells, without killing the latter. This phenomenon is called CD8 T-cell non-cytotoxic antiviral response (CNAR). CD4 T-cell numbers are usually maintained well in such persons, without developing symptoms of immune deficiency. We aim to understand how CD8 T-cells stop HIV replication in CD4 T-cells. In the proposed project, we will use a type of molecular scissors called CRISPR-Cas9 to cut and mutate the genes of HIV-infected CD4 T-cells. This pool of mutated CD4 T-cells will be grown with CD8 T-cells from i) elite HIV controllers, ii) non-elite-controlling persons with HIV, and/or iii) healthy individuals without HIV. If deletion of certain gene(s) in CD4 T-cells inhibits the antiviral activity of CD8 T-cells, we can infer involvement of these genes in the antiviral response. In this study, we propose to delete all 20,000 human genes in CD4 T-cells at once and examine their effect on the anti-viral activity of CD8 T-cells. The knowledge gained from these experiments is hoped to contribute towards a cure for HIV.


Genetic modification of HIV-specific T cell differentiation state to promote control of HIV
Rachel Rutishauser, MD PhD
UC San Francisco

HIV is an infection for which there is currently no cure. Many approaches to curing HIV seek to harness the power of anti-HIV T cells to specifically recognize and kill cells in the body that are infected with HIV. Because T-cells can become dysfunctional - or “exhausted” - during chronic HIV infection, we need to find approaches that prevent anti-HIV T-cells from losing their function. In this proposal, we will test cutting-edge approaches to overcome anti-HIV T-cell exhaustion that are concurrently being evaluated to improve T-cell-based therapies for cancer, another setting where T-cell exhaustion can hinder the efficacy of these treatments. Specifically, we will make anti-HIV T-cells (called CAR-T-cells, or chimeric antigen receptor T-cells) that we genetically engineer to not become exhausted by targeting the expression of genes that control T-cell exhaustion. We will target one gene that we have found gives anti-HIV T-cells the ability to proliferate robustly when they encounter HIV. Using CRISPR technology for gene editing, we will then target several other genes that we hypothesize may have a similar effect based on our preliminary studies in T-cells isolated from people with HIV who naturally control the infection and have highly functional anti-HIV T-cells. Finally, we are proposing to develop a novel system in the laboratory that will allow us to evaluate the function of anti-HIV CAR-T-cells in lymph nodes, the natural environment where HIV-infected cells persist in the body. Our studies will help us to identify and test new targets to improve the function of anti-HIV T-cells for HIV cure.


A molecular link between neuronal activity and CNS inflammation.
Sunnie Yoh, PhD
The Scripps Research Institute

Due to the advent of combination antiretroviral therapies (cART), systemic viral loads and morbidity rates in people leaving with HIV have been drastically reduced. Nonetheless, a significant portion of persons living with HIV exhibit neurocognitive disorders, which manifest in a broad spectrum from mild to severe conditions that include dementia or encephalitis. Several lines of evidence suggest that HIV continues to replicate at low levels in the central nervous system (CNS) even when cART suppresses systemic viremia. Other evidence suggests that persistent CNS inflammation, potentially induced by this low-level viral replication, may be a significant cause of HIV-1 associated neurocognitive disorders (HANDs). Phenotypic approaches to understanding the impact of HIV-associated neuroinflammation on CNS homeostasis is challenged by the fact that neuroinflammation is a concerted action of immune cells, vascular cells and neurons, whose responses can be inflammatory to anti-inflammatory simultaneously and whose outcome can be both neuroprotective and neuropathic.  Thus, we propose to identify molecular links among neuronal activity, CNS inflammation and neuropathological outcome in people living with HIV. We hypothesize that a master regulator of neuronal synaptic plasticity Arc (activity regulated cytoskeleton associated protein) modulates the inflammatory response to HIV infection in the CNS, and that this action potentially contributes to neuropathological outcomes in people living in HIV.

Basic Biomedical Discovery Initiative 2020

Microbiome Mechanisms in Substance Use and HIV Inflammation
Jennifer Fulcher, MD PhD

Despite the success of antiretroviral therapy in controlling HIV replication, chronic HIV infection causes persistent inflammation and increases risks of other health problems such as cardiovascular disease, bone disease, and cancer. These risks are even greater among substance using persons living with HIV, especially with stimulant use such as methamphetamine. It is important to understand what causes this chronic inflammation in order to develop better tools to treat substance use and HIV disease.

Many studies have shown a connection between the body’s commensal microbiota (also called the microbiome), mucosal immune system, and HIV-related inflammation. Substance use can also affect the body’s microbiota, and this may be one reason why HIV disease is worse among substance users. While the intestinal microbiome has been the focus of intense research, less is known about the role of the oral microbiome in health and disease. Studies examining the oral microbiome in the setting of HIV are limited and inconclusive. In non-HIV settings, the oral microbiome has been associated with increased risk of inflammation-related diseases such as cardiovascular disease. No published studies have examined the oral microbiome in methamphetamine users, despite known detrimental effects of methamphetamine on oral health. We intend to fill this gap in knowledge by examining the effects of methamphetamine use and HIV on the oral microbiome and resultant immune changes.

Our study will utilize a well-defined longitudinal cohort with archived saliva specimens and detailed substance use data for comprehensive microbiome analyses using metagenomics and novel analytic approaches. We will correlate the oral microbiome with systemic inflammation using immune biomarker analysis of paired blood specimens. Additional studies will use human oral mucosal tissue explants and mass cytometry to test relevant immune functions.

The data from this study will identify oral microbiome changes associated with HIV and/or methamphetamine use, and importantly will correlate these changes with immune and inflammatory outcomes. The goal of this work is to help better understand the complex relationships between substance use, the microbiome, and inflammation in HIV. The impact of the oral microbiome is an overlooked, yet potentially important, area for intervention. This is a critical step for developing better therapeutics.

Mechanism of Autophagy Modulation by HIV-1 Nef
James Hurley, PhD
University of California, Berkeley

The human immunodeficiency virus (HIV) infects human immune cells, avoids detection by the immune system, and causes acquired immune deficiency syndrome (AIDS). HIV encodes only 15 proteins, compared to more than 20,000 encoded by the human genome. HIV exerts profound effects on the cells it infects by virtue of its so-called accessory factors, whose job is to hijack our own cellular machinery. Over 400 human proteins are known to interact with HIV accessory factors.  One of these factors is the Nef protein. Patients infected with strains of HIV that have a defective version of Nef remain disease-free for years to decades, and are known as "long-term non-progressors". It would be highly desirable to have HIV Nef inhibitors that would have a similar effect in patients infected with normal HIV, however, the Nef protein does not have any one obvious point of vulnerability for us to attack.

Our laboratory uses structural biology and biochemistry to study how Nef interacts with human protein complexes, in search of sites that could be targeted to make Nef inhibitors. We have spent a number of years productively working out how Nef targets the cell process of endocytosis. Our lab also studies cellular self-eating, known as "autophagy". Autophagy is used to survive in starvation but can also be used by cells as a host defense against invading intracellular pathogens, which include HIV. Until now, little has been known about whether HIV is targeted by autophagy in the cell, and whether HIV has ways to fight back against autophagy, or even manipulate autophagy to its own ends. Our lab recently obtained some evidence that HIV Nef directly binds to and inhibits one of the main human protein complexes involved in autophagy. In this project, we will work out how this happens in enough detail to determine whether and how important this effect really is for HIV infection. If we are able to verify the effect is important for infection, the data we obtain will be the starting point for designing Nef inhibitors that reverse the effect and so help enable our own cell's defense to fight back against infection.

Using CRISPRi to Identify Host Genes Regulating HIV Latency
Zichong Li, PhD
The J. David Gladstone Institutes

After entering human CD4 T lymphocytes, HIV integrates within human DNA and immediately engages a variety of host genes. In most cells, viral replication occurs allowing dissemination of the virus. However, in some rare cells, the virus is repressed giving rise to a reservoir of latently infected cells. These latent proviruses are not affected by antiretroviral therapy (ART). However, if ART is withdrawn, virus present in the latent reservoir can reseed systemic viral infection. Currently, people living with HIV must take antiretroviral pills daily for their entire life to prevent viral rebound from the latent reservoir. Efforts to achieve a cure for HIV infection require a better understanding of the host genes involved in establishing and maintaining HIV latency.

This two-year Basic Biomedical Pilot Award from California HIV/AIDS Research Program seeks to identify host genes that repress HIV replication resulting in latently infected cells. In Aim 1, using a new technique termed CRISPR interference (CRISPRi), we will examine the potential repressive function of each of the 20,000 human protein-coding genes. Groups of these genes will be shut down and the cells assessed for a return of HIV expression. Isolation of the virus-producing cells will permit the generation of enriched CRISPRi libraries. Serial rounds of enrichment will allow us to home in on a set of latency-promoting genes.

Identification of these HIV repressors could lead to strategies for either purging or silencing the latent reservoir. In Aim 2, we will validate the function of these latency promoting genes in primary CD4 T cell models of HIV latency and in latently infected cells obtained from HIV-infected individuals on long term ART. We have already isolated four candidate latency promoting genes and are currently testing their function in the primary CD4 T cell models. Ultimately, small molecule inhibitors of these latency genes could form a new and exciting class of latency reversing agents. Conversely, activators of these cellular genes might be useful in block and lock strategies designed to permanently silence latent HIV proviruses.

Renal Effects of Hormones/Biomarkers in Transgender PrEP Patients
Nimish Patel, PharmD PhD
University of California San Diego, Skaggs School of Pharmacy & Pharmaceutical Sciences

Rates of newly diagnosed HIV infection are 3 times the national average in the transgender/non-binary (TGNB) population and nearly 14% of TGNB individuals are living with HIV infection. Currently, there are two commercially available drug products approved for preventing HIV: emtricitabine (F) in combination with either tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF). The use of these F/TDF or F/TAF to prevent HIV is commonly referred to as pre-exposure prophylaxis (PrEP). While both F/TDF and F/TAF are effective at preventing HIV, these medications are cleared by the kidneys and their dosing relies on accurate estimation of kidney function to initiate PrEP and monitor for side effects. The most prevalent method of estimating kidney function involves calculating creatinine clearance (CRCL) using the Cockcroft-Gault (CG) equation. The CG equation was developed over 40 years ago and only considers age, weight, sex and serum creatinine. It does not differentiate sex assigned at birth versus current gender identity, nor does it account for the use of hormones, such as testosterone or estrogen. This is important because hormones are very commonly used in the TGNB population.

We are interested in assessing the relatedness of using of hormones (tablets, injections, creams, etc), actual amount of hormones measured in blood (i.e. estradiol, free/total testosterone), different blood/urine biomarkers that predict kidney injury or function, drug levels of F/TDF and F/TAF in the blood and measured kidney function. Our hypothesis is that use of masculinizing hormones like testosterone and having higher levels of testosterone in the blood will be associated with decreased kidney function and higher PrEP drug levels.

We plan to assess a population of 40 TGNB adults who are taking F/TDF for PrEP and plan on switching to F/TAF. The study population will be comprised of  four groups: i) TGNB individuals assigned male at birth (MtF) taking estrogen, ii) MtF not taking estrogens, TGNB individuals assigned female at birth (FtM) taking testosterone  and iv) FtM not taking testosterone. Study participants will come in for two study visits: once while on F/TDF and again after starting F/TAF. At each visit, they will be given a small dose of iohexol (medication that will help determine how well the kidneys are functioning) and four blood and one urine sample will be collected.

This study will be among the first to refine the estimation of kidney function in TGNB individuals. We will determine if the accuracy of CRCL estimating equations can be improved by including the use of hormones, the presence of various kidney biomarkers, and gender identity. The study’s potential findings have broad implications for clinical practice beyond HIV prevention and include management of other medications for other health conditions that require an accurate understanding of kidney function in the TGNB population.

Single Cell Transcriptomics of Ex Vivo HIV-infected Cells
Sushama Telwatte, PhD

When immune cells targeted by HIV, such as CD4+ T cells, become latently-infected, they do not produce virus and are largely invisible to the immune system. However, this hidden infection is reversibly-silent, meaning that latently-infected cells cannot be cleared by the immune system and can reactivate once an individual ceases drug treatment. These latently-infected CD4+ T cells are thought to be the main barrier to HIV cure and likely also contribute to the reduced life expectancy and higher incidence of diseases that are observed even in individuals who are optimally treated with currently available drugs.

In order to develop better therapies for HIV, including those aimed at cure, it is essential to understand the mechanisms that determine whether an infected cell will produce virus (productively infected) or establish this silent, latent infection. A major goal of HIV cure research is understanding which human cellular proteins govern latent vs. productive infection, and whether there is a cellular gene signature that can identify and target latently-infected cells.

Most CD4+ T cells and most HIV-infected cells reside in lymphoid tissues, particularly the gut. Several challenges exist for studying HIV latency in vivo: (1) latently-infected CD4+ T cells are indistinguishable from uninfected cells and are rare; (2) exceedingly low RNA expression is emblematic of latently-infected cells; and (3) bulk cell analyses fail to provide the necessary resolution to study these cells. Also, most methods rely on T cell activation to detect latently-infected cells, which can cause dramatic changes to the cell and preclude study of the original cell state. Single cell analyses of the blood and gut are necessary to examine the precise mechanisms that underlie lifelong maintenance of HIV latency.

We propose novel single-cell approaches that are sensitive enough to detect the viral genome in single infected cells from HIV-infected individuals (with and without activation) and simultaneously characterize multiple key aspects of the infection (such as the proviral genome, viral transcriptome, and human transcriptome) to identify differences between latently-infected, productively-infected, and uninfected cells from the blood and gut. These studies will provide insight into the mechanisms that underlie the maintenance of HIV latency, which could help us to devise better strategies to target and eliminate HIV.

Negative Impact of Type I Interferon in HIV Infected Infants
Christel Uittenbogaart, MD

An infant’s immune system is functionally immature making it difficult to combat infections.  The immune system of infants infected with or exposed to HIV, is even more compromised as reflected by a higher incidence of morbidity and mortality due to infectious causes, including other virus infections.  This is in part related to the immaturity of immune cells that make antiviral proteins including Type I interferons (IFN). The main producers of IFN are the plasmacytoid dendritic cells (pDC).  It has been reported that neonatal peripheral blood and cord blood pDC produce lower levels of Interferon-α (IFN-α) after exposure to viruses such as human Cytomegalovirus (CMV) or Herpes Simplex virus 1 (HSV1) as compared to adult pDC. 

Type I IFN include several subtypes of IFN-α as well as IFN-β. The IFN-α subtypes have different functions.  For example IFN-α2, has been studied extensively for its antiviral effects in acute HIV infection in adults and was found to have only a minor effect on HIV replication.  In contrast, IFN-α14 and IFN-α8 do show anti-HIV activity.  However there is limited information on the impact of HIV infection, or exposure to HIV, on the production of different subtypes of IFN-α and IFN-β by cord blood pDC.  Furthermore, little is known about how HIV infection/exposure alters the expression of the Type I IFN receptors on cord blood mononuclear cells.

In the proposed research we will test our hypothesis that “Neonatal/cord blood pDC exposed to, or infected with, HIV produce IFN-α subtypes with low antiviral activity with negative consequences for antiviral responses”.  A decrease in IFN-α subtypes with antiviral activity has consequences for effective control of HIV in neonates born to HIV infected mothers and should therefore be examined.  In addition to differences in anti-HIV activity of the IFN-α subtypes, we need to investigate the differential impact of HIV infection on IFN-β as we found that IFN-α and IFN-β play distinct roles in HIV infection.

The proposed research is significant as it addresses a neglected facet of the immune response in HIV infection and could reveal new targets for therapeutic approaches to improve the control of HIV and secondary infections.  This proposal leverages the expertise of the lead PI and co-PIs in pDC biology and innate immunity, and the synergy among the laboratories will facilitate the investigation of this highly innovative and underexplored question.  

Bispecific Chimeric Antigen Receptors to Minimize HIV Escape
Otto Yang, MD

Chimeric antigen receptors (CARs) can be delivered to immune cells via gene therapy to retarget the immune system. This targeting is generally achieved by a portion of the CAR that is an antibody against the desired target. When that antibody portion binds its target, such as a particular protein on a cancer cell, the CAR activates the immune cell to attack the cell with the target. In recent years, CAR gene therapy has made remarkable inroads into treating some cancers.

Ironically, the very first CAR therapy was targeted against HIV, but abandoned after clinical trials about 20 years ago showed no obvious efficacy. Gene therapy was in its infancy at that time, and technical challenges probably contributed to this failure. Meanwhile, there have been significant technological advances making CAR gene delivery successful in cancer treatment. Scientists are now revisiting the possibility of trying CARs for HIV treatment again.

One significant issue is the remarkable ability of HIV to mutate. It generally easily adapts to avoid immunity including antibodies. This is an area that has yet received little attention in the CAR field, but will certainly be a barrier for CAR therapy for HIV, which are based on the ability of antibodies to bind the virus. Analogous to current drug therapies, it is likely that combinations of antibody targeting will be required for successful CAR.

This project will tackle this issue. It will utilize a novel antibody engineering technology to combine two different antibodies into HIV-targeted CARs, which will make them more resistant to viral escape because HIV would need to avoid two different antibodies simultaneously. Two antibodies may still not pose enough of a barrier to prevent viral escape, so the project will also test combinations of these dual-CARs. The overall goal is to identify combinations of CARs that will be able to contain HIV without allowing it to mutate and escape from immune control. This would be a key advance in applying CARs to the functional cure of HIV infection.

Innate Sensing of HIV-1 Infection by PQBP1/cGAS in Microglia
Sunnie Yoh, PhD
Sanford Burnham Prebys Medical Discovery Institute

Microglia cells are residential macrophages of the CNS and the key innate immune cells whose response dictates inflammatory states in the brain.  Aberrant microglial activation, including pathogenic infection, is considered as a key contributor to neurodegenerative diseases such as HIV-associated neurocognitive disorders (HANDs). In accordance with the current unmet need to understand the molecular details of HIV-1 induced neuroinflammation, we are interested in understanding the innate immune sensing of HIV-1 infection in microglia.

Previously, we and others have discovered that cyclic GMP-AMP synthetase (cGAS) is a key player of the innate immune response to HIV-1 infection in myeloid cells such as macrophages and dendritic cells.  Importantly, through siRNA screening, we identified polyglutamine track binding protein 1 (PQBP1) as an essential cofactor of the cGAS mediated innate immune sensing of HIV-1 infection. This application leverages a large body of preliminary data generated in HIV-1 infection sensing by PQBP1/cGAS complex in myeloid cells. Based on the functional similarity between blood macrophages and microglia, we hypothesize that an analogous mode of innate immune sensing of HIV-1 infection occurs in microglial cells.

The goal of this study is to establish the presence of the cGAS sensing of HIV-1 infection in microglia which will test a feasibility of future full-scale interrogation on this innate sensing circuits in the microglia.  Abundant clinical evidences on chronically infected patients including the ones with HIV-1 encephalitis indicate that microglial activation exist in a spectrum of activation status. Nonetheless, how the mechanisms regulating the differential microglial activation are largely veiled. If our hypothesis is proven such that the PQBP1/cGAS functions as a rate limiting step of the innate immune responses against HIV-1 infection and this step is regulated differently depending on the cellular context, our model can provide a molecular basis of the apparent diversity in microglial activation states during the course of HIV-1 infection.

Unraveling the interface between the cellular state and permissiveness to innate response will certainly provide insight into the aging-associated dysfunction of microglia whose phenotypes resembles immune senescence.  

Basic Biomedical Discovery Initiative 2016

CHRP selected nine investigators from various institutions across California as recipients of the 2016 Innovative, Developmental Exploratory Awards (IDEAs) in basic biomedical science. Learn more about these projects by clicking on the following link (Basic Biomedical Science Awards 2016).