Pilot Research Projects

PIs: Vivien Sheehan (Emory Pediatrics), Rama Amara (Emory Primate Center)

CRISPR/Cas9 Based In Vivo Gene Therapy for Sickle Cell Disease

Sickle cell disease (SCD) is the most common inherited blood disorder, affecting millions worldwide. Due to a mutation, an abnormal form of hemoglobin (Hemoglobin S) develops which tends to polymerize in blood red cells, causing them to change their shape (to "sickle"). Ninety-eight percent of the world's SCD population resides in low-resource countries. Half of the affected children die before the age of 5, and 95% by 18 years of age. Strategies to treat patient with SCD rely on increasing the amount of fetal hemoglobin (HbF) in these patients, a form of hemoglobin that will not lead to sickling. Gene-based therapy to achieve this goal is becoming available in some developed country but is financially and logistically out of reach in developing regions of the world.

The proposed work seeks to develop a new cost-effective CRISPR/Cas9 based in vivo gene-editing strategy by which the production of fetal hemoglobin will be induced by disrupting the erythroid binding site of the HbF inhibitor, BCL11A. This strategy will be tested in nonhuman primates, as they can produce HbF and are of sufficient size and similarity to humans. The experiments will involve injections of adenoviruses carrying the Cas9 and CRISPR guide RNA that targets the erythroid binding site of BCL11A, into the bone marrow.

Aim 1: Determine editing efficiency in vitro in primate immune cells.

Aim 2: Assess HbF induction in Rhesus macaques subjected to the proposed gene editing approach.

PIs: Michael Treadway (Emory Psychology), Vas Michopoulos (Emory Primate Center)

Social status determinants of maladaptive glutamate responses to stress in nonhuman primates

Anhedonia is a highly disabling feature of major depression. It may result from prolonged exposure to stress, perhaps related to alteration in glutamatergic transmission in the medial prefrontal cortex. In recent studies in human controls, glutamate in the medial frontal cortex is reduced in the presence of chronic stress, an adaptive glutamate response (AGR) that appears to be absent in patients with major depression. The goal of this pilot proposal is to examine whether such AGR responses are present in monkeys. If so, this would enable subsequent mechanistic R01 proposals to examine the biologic basis for the observed differences, and to develop new therapeutic strategies for treatment of anhedonia.

To determine whether the AGR occurs in monkeys, the brains of macaque monkeys will be imaged (in vivo) with MR spectroscopy (MRS), a magnetic resonance imaging sequence that is sensitive to the presence of glutamate. The study will focus on glutamate measurements in the medial frontal cortex. In addition, stress responses will be assessed by measurements of cortisol and other serum markers. The PIs will use their knowledge of the social hierarchy in macaque groups to study the chronic effects of psychosocial stress exposure on the AGR, because lower social rank is known to be associated with chronic stress. The study will compare stress responses in animals of low, medium, and high social rank. For an examination of the effects of acute stress in these groups, all animals will be scanned shortly after transportation from the Primate Center field station to the main center, and then again, 5-7 days later. Because the transportation from the field station is a stressful experience for the animal, the first scan will be considered an "acute stress scan", while the second scan is expected to reflect a control scan.

Aim 1: Determine whether the AGR is detectable using MRS in anesthetized animals.

Aim 2: Explore associations between serum markers of stress axis and the detected changes in the medial frontal cortex.

PIs: Tauras P. Vilgalys (University of Chicago), Vas Michopoulos (Emory Primate Center)

Genetic variation in the response to yellow fever in nonhuman primates

The COVID-19 pandemic is an obvious example for the fact humans can develop severe symptoms to zoonotic diseases that lead to only mild symptoms in their natural host. Similar examples exist for other diseases, some of which involve nonhuman primate species. The study of species differences in immunity, contrasting species that are sensitive to those that are only minimally affects may lead to insights into strategies to control infection. Recent studies established genetic variation is a key factor that influences immune responses and disease susceptibility.

The planned studies will investigate the transcriptomic response to yellow fever, a disease that leads to different outcomes in two monkey species, sooty mangabeys (Cercocebus atys) and rhesus macaques (Macaca mulatta). Sooty mangabeys are a natural host to yellow fever and develop only mild symptoms, while yellow fever infections in rhesus macaques are severe, similar to the response in humans. The

The planned in vitro studies will use infections of peripheral blood mononuclear cells (PBMCs) with yellow fever virus and subsequent single-cell RNA sequencing to generate a fine-grained picture of how genetic variation shapes gene expression during the immune response across the different cell types found in PBMCs, allowing the capture of critical interactions between immune cells. This study will provide preliminary results for future studies that will examine immune mechanisms that may underlie differences in the susceptibility to infectious diseases. This line of research is anticipated to have broad implications for understanding primate adaptation, host-pathogen evolution, and immune variation.

Aim 1: Characterize the immunogenetic response to yellow fever in nonhuman primates and compare the results to data obtained in human patients with yellow fever.

Aim 2: Identify genetic variation which affects the immune response to yellow fever.

Shannon Gourley, PhD

Single-Cell Profiling of Immune Activation After Early Life Adversity

Early life stress adversely affects health, increases the susceptibility to stress and negatively affects immune functions. Leveraging samples collected by researchers in the EPC Division of Developmental and Cognitive Neuroscience (DCN) at the center's Field Station, Dr. Gourley and her research team will identify how chronic social subordination stress in monkeys early in life results in individual differences in immune system functions. The team hypothesizes early life stress induces similar transcriptional changes in peripheral blood monocytes in rhesus macaques and mice, and the number and gene expression profiles of monocytes correlates with behavioral deficits in mice exposed to early life social adversity.

Aim 1: The research team will analyze cell-type specific immune functions in macaques, comparing animals that experienced chronic social subordination stress to others that did not experience such stress.

Aim 2: The research team will carry out similar studies in mice.

Susan P. Ribeiro, PhD

Dissecting the Mechanisms of SARS-COV2 Protection Induced by 3M-052+alum+RBD Vaccination in the Upper Respiratory Tract of Rhesus Macaques

The effectiveness of current COVID-19 vaccines varies from 60% to more than 90%. While vaccination reduces hospitalization, it does not avoid infection. Dr. Ribeiro's research team has developed a vaccine based on the receptor binding domain (RBD) of the SARS-CoV-2

spike protein formulated with alum, with or without the 3M-052, a TLR-7/8 agonist. They found adding 3M-052 to the vaccination in rhesus macaques significantly decreased virus replication in nasal swabs (thus, reduced infectivity), despite inducing similar titers of neutralizing antibody in the bronchioalveolar lavage fluid. This suggests the 3M-052 vaccination induces additional immune modulation in the upper respiratory tract, an effect that could be used to develop next-generation vaccination approaches.

Aim 1: The researchers will study vaccination-induced changes in gene signatures of immune cells in the upper respiratory tract.

Aim 2: The researchers will evaluate the lung microenvironment in which such gene signature changes occur.

Sudhir Pai Kasturi, PhD

Evaluating a new SARS-CoV-2 receptor binding domain (RBD) based COVID-19 vaccine with clinical alum and alum adsorbed 3M-052 adjuvants in rhesus macaques

Significance: COVID-19 is a new infectious disease in humans caused by a novel coronavirus (CoV) named SARS-CoV-2. The rapid global spread of SARS-CoV-2 infections highlights an urgent need for a vaccine and/or therapeutics to stem the pandemic.

Specific Aim: To evaluate a new SARS-CoV-2 receptor binding domain (RBD) based COVID-19 vaccine with clinical alum and alum adsorbed 3M-052 adjuvants in rhesus macaques

Mirko Paiardini, PhD

Determining SARS-CoV-2 pathogenesis, viral dynamics, and immune responses in nonhuman primate models

Significance: Little is known regarding the viral pathogenesis and immune response to SARS-CoV-2. Given the devastation the disease is causing worldwide, it is imperative to elucidate the viral pathogenesis and early immune responses that may inform the treatment of infected individuals, identification of correlates of protection, and design of therapeutics and vaccines.

Specific Aim #1: To characterize the breadth of the early innate and adaptive immune response and pathogenesis of SARSCoV-2 infection systemically and localized within the respiratory tract.

Specific Aim #2: To assess the replication kinetics and tissue localization of SARS-CoV-2. Thomas Vanderford, PhD, Director of the EPC Virology Core will lead this component of the pilot project.