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Human Antibody Response to Flaviviruses and SARS-Coronavirus-2
Get PDFThe last decade has seen a number of viral epidemics and global pandemic. Increased global connectivity makes the likelihood of future mass outbreaks ever greater. Understanding the adaptive immune response to these pathogens will expand basic understanding of viral immunity mechanisms and can help inform vaccine and therapeutic development. This thesis describes the characterization of the human antibody response to several important viral pathogens, all of which are either emerging pathogens posing significant public health burdens or are already responsible for epidemic outbreaks. These include tick-borne encephalitis virus (TBEV), Powassan virus (POWV), Zika virus (ZIKV) and severe acute respiratory syndrome coronavirus (SARS-CoV-2). The first part of this thesis focuses on encephalitis-causing tick-borne flaviviruses, specifically TBEV and POWV. Both TBEV and POWV are emerging human pathogens that cause potentially fatal disease; there are no disease-specific treatments. Mouse monoclonal antibodies are protective against TBEV and POWV, but little is known about the human antibody response to active infection. Although 10,000 to 20,000 cases of TBEV infection are reported per year, study of the immune response to POWV is additionally complicated by the paucity of confirmed cases. Here I report on the human neutralizing antibody response to TBEV in a cohort of infected and vaccinated individuals, and to POWV in a cohort of infected individuals. Memory B cells expressing anti- TBEV envelope domain-III (EDIII) antibodies were isolated using a combination of single-cell sorting and nested polymerase chain reaction (PCR). Expanded clones of memory B cells expressing closely related anti-EDIII antibodies were found in both groups of volunteers. Antibodies representative of clonal and unique sequences found in TBEV-infected and -vaccinated donors were expressed as monoclonals and characterized functionally. In reporter virus particle (RVP) neutralization assays, the most potently neutralizing antibodies with IC50s below 1ng/mL were found only in convalescent donors. Neutralization assays using authentic TBEV in vitro confirmed potent neutralization activity in at least the four antibodies tested. Select antibodies also neutralized RVPs representative of other emerging tick-borne flaviviruses including Langat, louping ill, Omsk hemorrhagic fever, Kyasanur forest disease, and Powassan viruses. Prophylactic or early therapeutic antibody administration was effective at low doses in mice that were lethally infected with TBEV. Shifting focus to the ongoing SARS-CoV-2 pandemic, this virus continues to be a global problem in part because of the emergence of variants of concern that evade neutralization by antibodies elicited by Wuhan-Hu-1 infection or vaccination. I report on the human neutralizing antibody and memory responses to the Gamma variant in a cohort of hospitalized individuals from Manaus, Brazil. Plasma from infected individuals potently neutralized viruses pseudotyped with the Gamma SARS-CoV-2 spike protein, but neutralizing activity against Wuhan-Hu-1, Beta, Delta, or Omicron was significantly lower. Memory B cells expressing anti-Gamma receptor binding domain (RBD) antibodies were again isolated using a combination of single-cell sorting and nested polymerase chain reaction (PCR). Monoclonal antibodies representative of clonal and unique antibody sequences were expressed and characterized functionally. In an in vitro neutralization assay using pseudovirus, antibodies from Gamma-infected individuals were shown to neutralize Gamma and Beta pseudoviruses more effectively than Wuhan-Hu-1. Sixty-nine % and 34% of Gamma-neutralizing antibodies failed to neutralize Delta or Wuhan-Hu-1. In contrast to class 1 and 2 antibodies that dominate the response to Wuhan-Hu-1 or Beta, 43% of antibodies elicited by Gamma infection recognized Class 3 epitopes. These results have implications for variant-specific vaccines, suggesting that these strategies would have limited efficacy. In the course of characterization, specific antibodies with unique properties were identified. In the final part of this thesis, I return to these antibodies to investigate them in more detail. They include antibody T025, which was shown to be efficacious at preventing disease in an in vivo model of infection. Structural analysis revealed a conserved epitope near the lateral ridge of EDIII adjoining the EDI-EDIII hinge region. Antibody T036, also from a TBEV-infected donor, was shown separately to enhance RVP and authentic virus infection in vitro in a Fc receptor-independent manner. Enhancement could be ablated by addition of 4G2, a mouse anti-fusion loop monoclonal antibody. T036 was shown to bind a cryptic epitope on the TBEV EDIII, suggesting that T036 binds the virion during or after conformational change of the E protein. Separately, Z039 and Z015 had been shown in previous studies of anti-ZIKV EDIII antibodies in ZIKV-exposed cohorts to exhibit unexpectedly broad cross-reactivity. Z039 and Z015 were confirmed to bind ZIKV EDIII, as well as Dengue virus 1-4 EDIIIs, and yellow fever virus and West Nile virus EDIIIs, respectively. in vitro neutralization assays using authentic virus confirmed corresponding patterns of antibody-mediated neutralization. Z039 was shown to recognize an epitope on the flavivirus EDIII similar to that of Z021, an anti-Zika antibody with comparatively limited cross-reactivity, although Z039 appears to make contacts with the EDIII backbone which may partially explain its breadth of activity. Z015, on the other hand, does not compete with Z021 or Z004 and recognizes a distinct but cryptic epitope on the yellow fever virus EDIII
Peptidergic Signaling Controls the Dynamics of Sickness Behavior in Caenorhabditis Elegans
Get PDFDisease is accompanied by modifications to host behavior that promote recovery and survival. The neural and molecular mechanisms that rule the switch to a sickness behavioral state are an active area of research. One established model for sickness behavior is the interaction between Caenorhabditis elegans and the pathogenic bacteria Pseudomonas aeruginosa PA14. After a few hours of exposure to and infection by PA14, C. elegans develops a sickness-like behavior termed pathogen avoidance. Here I identify neuronal signals generated during the infection and associate them with antagonistic circuits that shape the host behavior. Using an unbiased cell-directed neuropeptide screen, I show that the AVK neurons release FMRFamide-like FLP-1 neuropeptides during infection to drive avoidance of pathogenic bacteria. Manipulations that increase or decrease AVK signaling accelerate or delay pathogen avoidance, respectively, implying an instructive role in the behavioral decision. FLP-1 neuropeptides act on the G-protein-coupled receptor DMSR-7 which drives pathogen avoidance by inhibiting tyraminergic and octopaminergic signaling from neurons that play multiple roles in the pathogen response. My results demonstrate that antagonism between neuromodulatory systems results in graded transitions between alternative behavioral states
Biased Constitutive Activity in the Uveal Melanoma Oncogene CYSLTR2 is Unique in CYSLTR2 Germline and Pan-Cancer Human Variome
Get PDFUveal melanoma is the most common eye cancer in adults and is clinically and genetically distinct from skin cutaneous melanoma. In a subset of cases, the oncogenic driver is an activating mutation in CYSLTR2, the gene encoding the G protein-coupled receptor (GPCR) cysteinylleukotriene receptor 2. The mutant CYSLTR2 encodes for CysLTR2-L129Q receptor, with the substitution of Leu to Gln at position 129 (3.43). The ability of CysLTR2-L129Q to cause malignant transformation has been hypothesized to result from constitutive activity, but how the receptor could escape desensitization is unknown. In this work, we characterized the functional properties of CysLTR2-L129Q. CysLTR2 signals through the Gq/11/PLC-β pathways, so using a homogenous time resolved fluorescence (HTRF) IP1 accumulation assay, we show that CysLTR2-L129Q is a constitutively active mutant that strongly drives Gq/11 signaling pathways. However, CysLTR2-L129Q only poorly recruits β-arrestin as shown by a bioluminescence resonance energy transfer 2 (BRET2) based β-arrestin recruitment assay. Using a modified Slack-Hall operational model, we quantified the constitutive activity for both pathways and conclude that CysLTR2-L129Q displays profound signaling bias for Gq/11 signaling pathways while escaping β-arrestin-mediated downregulation. CYSLTR2 is the first known example of a GPCR driver oncogene that encodes a highly biased constitutively active mutant receptor. These results provide new insights into the mechanism of CysLTR2-L129Q oncoprotein signaling and suggest CYSLTR2 as a promising potential therapeutic target in uveal melanoma. Furthermore, we learned that CysLTR2 is a significantly mutated GPCR in several other cancers as well. We identified \u3e100 CYSLTR2 missense variants of unknown significance (VUS) in human cancer genomes from available cancer databases, as well as another \u3e100 CYSLTR2 single-nucleotide polymorphisms (SNPs) from exome sequence data. Here, we introduce a proof-of-concept, experimental, activity-based profiling pipeline to systematically assess the mutational landscape of CYSLTR2. We use a single transfection mixture of receptor-encoding DNA and HEK293T cells is used to characterize all variants for expression level, basal and agonist-stimulated G protein signaling, and basal and agonist-stimulated β-arrestin recruitment. The CysLTR2-L129Q mutation causing uveal melanoma has a unique phenotype among all cancer-associated variants. It is highly constitutively active with gain-of-function (GoF) in basal Gq/11-PLC-β signaling and loss-of-function (LoF) in agonist-dependent signaling, while only poorly recruiting β-arrestin. Furthermore, we found that about 21% of the variants show no detectable activity and are basically indistinguishable from mock-transfected controls, suggesting that a large portion of these mutations are damaging. A further 21% lose 50% of activity as normalized to WT (100%), and another ten percent are nonsense and frameshift variants. This means that about 50% of total somatic mutations of CYSLTR2 have a LoF phenotype, which points to a tumor suppressor function following the famous 20/20 rule
Tumor Suppression by a TRNA Synthetase
Get PDFDespite substantial progress in treatment, breast cancer remains a leading cause of cancer mortality among women. During malignant transformation, healthy mammary cells must bypass tumor suppressive checkpoints and activate pro-growth pathways. Enhanced protein translation is one such hallmark of tumor transformation and cancer progression; many oncogenes promote translation by driving synthesis and activity of translational machinery. In the first part of this thesis, we report the surprising finding that leucyl-tRNA synthetase (LARS), a tRNA synthetase responsible for ligating leucine to corresponding leucyl-tRNAs, becomes strongly repressed during mammary cell transformation and in breast cancer. Monoallelic genetic inactivation of LARS in mouse mammary glands enhanced tumor formation and proliferation. LARS repression reduced the abundance of specific leucyl-tRNA isoacceptors tRNA-LeuCAG, tRNA-LeuAAG and tRNA-LeuTAG. This repression resulted in decreased leucine codon-dependent translation and expression of key leucine-rich, growth suppressive genes. We uncovered two candidate tumor suppressor genes, EMP3 and GGT5, that act downstream of LARS. This work uncovers a tumor suppressive tRNA synthetase and reveals that its repression, along with its downstream tRNAs, can modulate translation to enhance breast tumorigenesis and proliferation in a codon-dependent manner. Modulation of translation by tRNAs and their associated factors can affect not only cancer transformation, but also normal cell physiology. In the second half of this thesis, we describe work on mammary epithelial cells which, in the context of oxidative stress, exhibited a stressinduced tRNA fragmentation response. Though tRNA fragmentation is classically not thought to occur at high enough levels to deplete mature tRNA pools, we demonstrated that tyrosyl-tRNA fragmentation depleted mature tRNA-TyrGUA following 24 hours of oxidative stress. tRNATyrGUA depletion was sufficient to reduce translation and expression of growth-promoting, tyrosine-enriched transcripts in a codon-dependent manner, contributing to the restriction of cell growth in response to stress. We further demonstrated that the tRNA fragment, tRF-TyrGUA, was generated in a DIS3L2 exoribonuclease-dependent manner and acted in trans with RNA binding proteins, including HNRNPA1, to inhibit HNRNPA1-mediated transcript destabilization. These findings uncover and characterize a second adaptive, codon-based regulatory response that can contribute to cellular stress responses in physiology and disease
Ras Drives Malignancy Through Stem Cell Crosstalk with the Microenvironment
Get PDFSquamous cell carcinomas (SCCs) are triggered by marked elevation of RAS/MAPK signaling and progression from benign papilloma to invasive malignancy. A subset of tumor-initiating basal progenitors, the cancer stem cells, obtain increased resistance to chemo and immunotherapy along this path. However, the distribution and changes in cancer stem cells during progression from a benign state to an invasive SCC remain elusive. Here we show that following HRASG12V activation, cancer stem cells rewire their gene expression program and trigger self-propelling, aberrant signaling crosstalk with their tissue microenvironment that drives their malignant progression. Surprisingly, the non-genetic, dynamic cascade of crosstalk involves pathways often mutated in advanced metastatic SCCs with a high mutational burden. Coupling our clonal skin HRASG12V model with single-cell transcriptomics, chromatin-landscaping, lentiviral reporters and lineage-tracing, we show that the aberrant cancer stem cell-microenvironment crosstalk creates conditions ripe for hijacking leptin receptor (LEPR)-signaling, which in turn launches downstream PI3K-AKT-mTOR signaling at the benign-malignant transition. By functionally interrogating each step in this pathway, we unravel how dynamic temporal crosstalk with the microenvironment and orchestrated by the stem cells, profoundly fuels this path to malignancy. This discovery provides new insights into the path to malignancy and suggests broad implications for cancer therapeutics
Library’s First Bookplate
No full textLibrary’s first bookplate replicating the Institute’s original logo, 1904
The first logo (seal) designed in 1904 by Francis Scott King was featuring Aesculapius (the Greek God of medicine) with a caduceus standing near small crossed alembics. It was in use for fifty years until the change in the Institute’s name in 1954 brought forth a need for a change in the traditional seal. It was decided to re-design it completely. Rudolph Ruzicka, of Boston, Massachusetts, a distinguished American artist, and engraver, was commissioned to the design. He determined that an almost purely typographical design would be superior to any of the rather strained and formal symbols that might be used. He retained and indeed made dominant, the original motto Pro Bono Humani Generis, which has appeared almost microscopically on the first Institute’s seal.https://digitalcommons.rockefeller.edu/objects-tell-stories/1003/thumbnail.jp
American Flad Displayed During the First Convocation
No full textThis flag (donated by Julie Francis) was displayed in 1959 when the first degrees were conferred by President Detlev Bronk. In the background is the photograph of the first graduating class.
In 1954, The Rockefeller Institute assumed the status of a graduate university, extending the scope of this educational function to include work at the doctoral level for a carefully selected group of young men and women who display unusual potential for contributing to the nation’s scientific progress. The first Ph.D. degrees were awarded in 1959. In 1965, the institution’s name was officially changed to The Rockefeller University.https://digitalcommons.rockefeller.edu/objects-tell-stories/1021/thumbnail.jp
Convocation 1961
No full textProgram of convocation for conferring degrees, 16 June 1961https://digitalcommons.rockefeller.edu/objects-tell-stories/1022/thumbnail.jp
Dr. Jules Hirsch’s Stethoscope
No full textDr. Jules Hirsch’s stethoscope, circa 1960s
Dr. Jules Hirsch (1927-2015) was a leader in the study of human metabolism mainly in the area of lipids and obesity. His research at The Rockefeller Hospital helped establish the mechanism of obesity and lipids metabolism. Dr. Hirsch joined Rockefeller´s faculty in 1954 and remained there for the rest of his career. Hirsch’s research helped to support the idea of dynamic interactions between diet, physical activity, general metabolism, and obesity. At that time most scientists considered adipose tissue to be biologically inert such as a passive insulator in which the body reserved energy in the form of triglycerides. The Rockefeller community remembers Dr. Hirsch as extremely intelligent and generous, with a tremendous variety of interests. He loved all living things.
See also Jules Hirsch Memoriam, 1927-2015https://digitalcommons.rockefeller.edu/objects-tell-stories/1027/thumbnail.jp
A Garland for President Bronk, 1968
No full textA Garland for President Bronk, 1968
With the inscription To Detlev W. Bronk, scientist, statesman, educator, successor to Simon Flexner and Herbert S. Gasser and first President of The Rockefeller University from administrative staff with devoted friendship and warm admiration.
In 1953, with the election of Dr. Detlev Bronk (1897-1975) to the newly created office of the President, an extensive reorganization of the Rockefeller Institute took place. The Rockefeller charter was amended by the University of the State of New York and given authority to grant doctoral degrees; in 1965, the name was changed officially to The Rockefeller University. Under Dr. Bronk’s guidance, the Rockefeller established a graduate student body and expanded research and teaching activities from the life sciences to physics, mathematics, philosophy, and psychology. Nine new buildings have been erected and additional land has been acquired for future expansion. In place of an individual, and oftentimes isolated, laboratories headed by senior scientists, Detlev Bronk created a campus complete with students, dormitories, and even a faculty club.https://digitalcommons.rockefeller.edu/objects-tell-stories/1026/thumbnail.jp