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Basic Biomedical Discovery Initiative 2019

These pilot awards support early-stage laboratory exploration aimed at understanding mechanisms of HIV prevention, treatment, or cure at the cellular or subcellular level. Each project consists of basic biomedical HIV research that is highly innovative, addresses a significant question or barrier in HIV science, and shows clear promise to yield findings that can serve as a basis for compelling studies of larger magnitude.
Microbiome Mechanisms in Substance Use and HIV Inflammation
Jennifer Fulcher, MD PhD
UCLA

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
UCSF

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
UCLA

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
UCLA

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.