Eva Harris, Professor

Closed (1) Protection from dengue pathogenesis mediated by anti-NS1 antibodies

Closed. This professor is continuing with Spring 2021 apprentices on this project; no new apprentices needed for Fall 2021.

With 3.6 billion people living at risk of infection, and approximately 400 million infections and 96 million cases per year, dengue is the most prevalent mosquito-borne viral illness. The disease spectrum includes dengue fever, characterized by debilitating symptoms such as high fever and myalgia; and dengue hemorrhagic fever/dengue shock syndrome, with life-threatening manifestations stemming from vascular leakage that leads to shock. Dengue virus (DENV) nonstructural protein 1 (NS1) circulates in patients’ blood during acute infection, and high levels of NS1 are associated with severe disease. However, it was not until recently that our laboratory demonstrated that NS1 directly triggers endothelial permeability and vascular leak. Further, we also showed that the pathogenic effects of NS1 can be blocked by NS1-immune polyclonal mouse serum or by monoclonal antibodies (mAbs) to NS1, and that immunization of mice with NS1 protects against lethal DENV challenge. Currently, our goal is to establish whether certain domains of NS1 have a direct role in pathogenesis and/or protection. Using molecular genetics and in vitro approaches, we determined that glycosylation of specific NS1 amino acids mediates NS1-induced hyperpermeability and glycocalyx dysfunction, while residues in other NS1 regions are critical for engaging innate immunity receptors. With a combination of immunological and murine in vivo assays, we will now map and assess the therapeutic capacity of mAbs against NS1 regions involved in pathogenesis and protection.

RECOMMENDED READING
1. Beatty PR, Puerta-Guardo H, Killingbeck SS, Glasner DR, Hopkins K, and Harris E. Dengue virus NS1 triggers endothelial permeability and vascular leak that is prevented by NS1 vaccination. Sci Transl Med. 2015;7(304):304ra141.
2. Orozco S, Schmid MA, Parameswasan P, Lachica R, Henn MR, Beatty PR, Harris E. Characterization of a model of lethal dengue virus 2 infection in C57BL/6 mice deficient in the interferon-alpha/beta receptor. J Gen Virol 93, 93:2152-2157 (2012).
3. Puerta-Guardo H, Glasner DR, and Harris E. Dengue Virus NS1 Disrupts the Endothelial Glycocalyx, Leading to Hyperpermeability. PLoS Pathog. 2016;12(7):e1005738.


The goal of this project is to characterize the specificity and in vivo protective capacity of anti-NS1 mAbs. Training will be focused on learning enzyme-linked immunosorbent assays (ELISA) and performing passive transfer experiments in mice. However, it is also expected that the URAP apprentice will learn how maintain animal breeding colonies and to handle, wean and genotype mice. Other skills to be learned comprise mouse inoculations through various routes, blood collection and basic cell culture techniques. The URAP apprentice will work with and under the supervision of Dr. Scott Biering in Dr. Eva Harris’ laboratory. A commitment of at least 12 hours per week is expected.

Day-to-day supervisor for this project: Scott Biering, Post-Doc

Qualifications: Applicant should be a sophomore level or higher. Experience with animal handling is highly preferred and a strong interest in immunology and infectious disease is desirable. Additional requirements include organizational skills, attention to detail and high motivation and enthusiasm. Working knowledge of Excel is a plus.

Weekly Hours: 12 or more hours

Related website: https://www.harrisresearchprogram.org

Closed (2) The interplay between dengue virus and the human immune system

Closed. This professor is continuing with Spring 2021 apprentices on this project; no new apprentices needed for Fall 2021.

The human immune system must be able to recognize and eliminate pathogens through frontline innate and adaptive responses. In turn, medically relevant pathogens, such as dengue virus (DENV: Flaviviridae, Flavivirus), have developed mechanisms to overcome immune recognition, allowing virus replication and dissemination. Further, an unbalanced immune response can be harmful to the host and contribute to disease severity. Here, we seek to explore two questions: i) how dengue virus escapes innate immune recognition; ii) how adaptive responses (e.g., anti-dengue antibodies) can correlate with disease progression.
For the first goal, we will investigate DENV non-structural protein 5 (NS5). NS5 is the largest viral protein, containing two domains: an RNA-dependent RNA polymerase and a methyltransferase (MTase) domain. The MTase domain is thought to contribute to viral RNA 5’-capping, which would allow viral genome translation while avoiding innate immune recognition from RNA cytosolic receptors. One possibility is that 5’-capping masks the 5’-ppp present in the viral genome that would otherwise trigger the cytosolic sensor RIG-I. RIG-I can sense 5’-ppp-bearing double-stranded RNAs leading to a signalling cascade that induces potent antiviral cytokines such as type I interferons (IFN I). Hence, our main hypothesis is that DENV NS5 can block RIG-I activation through its MTase domain. To address this, we will investigate flaviviral conserved residues within the MTase domain to dissect NS5’s ability to generate 5’-capped RNAs and induce IFN I through RIG-I sensing. Our main model will be an infectious clone of DENV-3, its MTase mutants, and cell lines that have been rendered knockout for RIG-I-like receptors.
The student will also have the chance to participate in our second project aimed at exploring the Fc functions of anti-DENV antibodies. Classically, disease protection and vaccine responses have been evaluated through antibody titers and neutralization potential. However, these two features alone have been unable to completely inform correlates of immune protection for several infectious agents (e.g., HIV, Ebola virus, dengue virus). In turn, antibodies exert additional functions that are dependent on the antibody constant region (Fc) and its interaction with innate immune cells (e.g., monocytes, macrophages, NK cells, neutrophils). Our team is currently exploring correlates of protection by profiling the Fc biophysical features (e.g., Fc-receptor interactions, affinity, avidity, isotyping) and functions (e.g., phagocytosis, cytotoxicity, complement activation) of polyclonal serum antibodies from our dengue pediatric cohort study in Nicaragua. Initial results might lead to mechanistic studies to explore how anti-dengue antibodies can play a dual role in disease severity and protection.

The URAP student will work under the direct supervision of a postdoctoral researcher in Dr. Eva Harris’ laboratory, Dr. Gregorio Dias, and be expected to work a minimum of 12-15 hours per week and commit to at least one year, ideally including the summer of 2020. This requires regular work on weekdays that may extend beyond after 5pm and occasional work on weekends or as arranged with the postdoctoral researcher. Skills learned will include but not be limited to cell culture and molecular biology techniques. The URAP student is also expected to attend weekly research meetings and maintain detailed records of the work performed. The student is expected to complete the assignments in a timely manner, maintain open communication with other members of the research group and with the research coordinator, ask questions when help or guidance is needed, and actively ensure (through communicating with the research coordinator) that he/she is getting the experience expected from the URAP program.

Day-to-day supervisor for this project: Dr. Gregorio Dias, Post-Doc

Qualifications: The applicant should be a sophomore level or higher, majoring in immunology, genetics, microbiology, or cell biology. Experience with sterile technique, cell culture, and infectious agents is preferred. This project requires a strong interest in infectious disease. Other requirements are enthusiasm, high motivation, attention to detail, a passion for science, and a desire to develop independent thinking. The applicant should be willing to work as a part of a team; be a self-starter, organized and have a high degree of attention to detail; and be committed to the project for the academic year (plus one summer).

Weekly Hours: 12 or more hours

Related website: https://www.harrisresearchprogram.org

Closed (3) Investigating molecular mechanisms of dengue virus NS1 disruption of the glycocalyx-like layer of human endothelial cells

Closed. This professor is continuing with Spring 2021 apprentices on this project; no new apprentices needed for Fall 2021.

Previous studies in the Harris laboratory have demonstrated the ability of the secreted flaviviral protein, nonstructural protein 1 (NS1), to induce both hyperpermeability in vitro and vascular leak in vivo, both mediated by the disruption of endothelial glycocalayx components. Additional work has demonstrated that distinct flavivirus NS1 proteins bind differentially to glycans, particularly heparan sulfate and chondroitan sulfate, on the cell surface. Given these findings, this project aims to investigate NS1 interactions and binding to candidate glycosaminoglycans (heparan sulfate, chondroitin sulfate, hyaluronic acid, sialic acid) in key human endothelial cells (lung, brain, umbilical cord, liver) while further characterizing the nuances and tropism of binding interactions among West Nile virus (WNV), Zika virus (ZIKV), dengue virus (DENV), and Japanese Encephalitis virus (JEV) NS1 proteins to candidate GAGs in each endothelial cell line. These data will provide insight into the mechanisms of flavivirus pathogenesis. The Specific Aims of this project are as follows:
1) To assess candidate GAG expression and magnitude in various human endothelial cell lines using IFA microscopy.
2) To determine the modulation of GAG expression after enzymatic treatment using IFA microscopy.
3) To assess how enzymatic cleavage of candidate GAGs in key endothelial tissues affects the ability of flavivirus NS1 to bind to the cell surface.

The URAP student will work under the direct supervision of a postdoctoral researcher in Dr. Eva Harris’ laboratory, Dr. Scott Biering, and be expected to work a minimum of 12-15 hours per week and commit to at least one year, ideally including the summer of 2019. This requires regular work on weekdays that may extend beyond after 5pm and occasional work on weekends or as arranged with the postdoctoral researcher. Skills learned will include but not be limited to cell culture and immunofluorescence microscopy. The URAP student is also expected to attend weekly research meetings and maintain detailed records of the work performed. The student is expected to complete the assignments in a timely manner, maintain open communication with other members of the research group and with the research coordinator, ask questions when she needs help or guidance, and actively ensure (through communicating with the research coordinator) that she is getting the experience she expects from the URAP program.

Day-to-day supervisor for this project: Francielle Tramontini, Post-Doc

Qualifications: The apprentice must be at a sophomore or junior level, and have a strong interest in molecular biology, biochemistry, and infectious disease. Experience with molecular biology and biochemistry is preferred. Other requisites are enthusiasm, high motivation, and the desire to develop independent thinking.

Weekly Hours: 12 or more hours

Related website: https://www.harrisresearchprogram.org

Closed (4) Evaluation of in vitro and in vivo efficacy of glycan-based compounds against flavivirus endothelial permeability & vascular leak

Closed. This professor is continuing with Spring 2021 apprentices on this project; no new apprentices needed for Fall 2021.

Dengue (DENV) and Zika (ZIKV) flaviruses are mosquito-borne viruses that are major medical and public health problems worldwide. DENV causes the most prevalent mosquito-borne viral disease of humans, and severe cases manifesting vascular leakage can be fatal. Nonstructural protein 1 (NS1) is a flaviviral protein that participates in viral RNA replication and in its secreted form plays important roles in host immune evasion and viral pathogenesis. We and others recently described novel roles for NS1 in directly triggering endothelial barrier dysfunction and inducing inflammatory cytokine production from human immune cells, contributing to vascular leak in vivo. Here, we will evaluate the in vitro and in vivo efficacy of glycans against NS1-mediated pathogenesis, as well as against DENV and ZIKV infection in vivo. The Harris lab has developed multiple methods to study DENV and ZIKV pathogenesis based on characterization of NS1-induced endothelial barrier dysfunction in vitro (e.g., hyperpermeability, disruption of the endothelial glycocalyx-like layer [EGL]) and in vivo, using murine models of virus- and NS1-induced disease with vascular leakage. nds inhibit NS1-induced EGL degradation and vascular leakage in vivo as well as viral infection and have potential as novel treatment modalities for dengue and Zika. We will select the most promising inhibitor(s) of NS1-induced pathophysiological pathways of DENV and ZIKV based on prevention of endothelial dysfunction in vitro. We will screen 27 glycans (including sulfated and non-sulfates) for their ability to prevent NS1-induced hyperpermeability. Next, we will investigate the in vitro mechanism of action of prevention of endothelial dysfunction by the selected compounds. Finally, we will evaluate the therapeutic potential of the most active compounds against DENV and ZIKV NS1- and virus-induced vascular leak, morbidity, and mortality in vivo. Overall, this addresses a critical need, identifying novel therapeutic strategies against two major flaviviral diseases, by developing glycan-based compounds that target both the virus and pathophysiological consequences of infection.


The URAP student will work under the direct supervision of a postdoctoral researcher in Dr. Eva Harris’ laboratory, Dr. Francielle Tramontini, and be expected to work a minimum of 12-15 hours per week and commit to at least one year, ideally including the summer of 2020. This requires regular work on weekdays that may extend beyond after 5pm and occasional work on weekends or as arranged with the postdoctoral researcher. Skills learned will include but not be limited to cell culture, ELISA, and immunofluorescence microscopy. The URAP student is also expected to attend weekly research meetings and maintain detailed records of the work performed. The student is expected to complete the assignments in a timely manner, maintain open communication with other members of the research group and with the research coordinator, ask questions when he/she needs help or guidance, and actively ensure (through communicating with the research coordinator) that he/she is getting the experience expected from the URAP program., Post-Doc

Qualifications: The apprentice must be at a sophomore or junior level, and have a strong interest in cell biology and infectious disease. Experience with sterile technique, cell culture, and microscopy is preferred. Other requisites are being highly motivated and having an enthusiastic scientific spirit and the desire to develop independent thinking.

Weekly Hours: 12 or more hours

Related website: https://www.harrisresearchprogram.org