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Article: Analysis of SARS-CoV-2 Variant-Specific Serum Antibody Post-Vaccination Utilizing Immortalized Human Hepatocyte-Like Cells (HLC) to Assess Development of Immunity.

Collins, Daniel P / Steer, Clifford J

Hepatic medicine : evidence and research

2023 Volume 15, Page(s) 221–231

Abstract: Background: Our previous studies demonstrated that SARS-CoV-2 spike protein could bind to primary hepatocytes and immortalized Hepatocyte-like cells (HLC) via the asialoglycoprotein receptor-1 (ASGR-1). The binding of biotinylated spike protein could be ...

Abstract	Background: Our previous studies demonstrated that SARS-CoV-2 spike protein could bind to primary hepatocytes and immortalized Hepatocyte-like cells (HLC) via the asialoglycoprotein receptor-1 (ASGR-1). The binding of biotinylated spike protein could be inhibited by Spike-neutralizing monoclonal antibodies, anti-ASGR-1 antibodies and unlabeled spike protein. The cells were unable to bind Spike S1 and Spike S1 was incapable of blocking labeled Spike protein, suggesting that the Receptor Binding Domain (RBD) was not involved in the binding event. This study was done to investigate the utility of these cells and immortalized alveolar type 2-like (AT-2) cells in studying the development of variant-specific antibodies post-vaccination. Methods: Serum was collected from 10 individuals pre- and post-vaccination with the J&J, Moderna or Pfizer vaccines. The serum samples were quantified for variant-specific antibodies in a flow cytometry-based immunofluorescent assay utilizing beads coated with biotinylated variant spike proteins. Inhibition of spike protein binding to HLC and AT-2 cells by donor serum was analyzed by immunofluorescent confocal analysis. Results: All variant spike proteins bound to HLC and AT-2 cells. Post-vaccination serum samples demonstrated increases of SARS-CoV-2 antibody levels from 2 weeks to 2.5 months post-vaccination with associated increased spike-blocking capacity. It was also demonstrated that vaccination with all the available vaccines stimulated antibodies that inhibited binding of all the available variant spike proteins to both HLC and AT-2 cells. Conclusion: HLC, along with AT-2 cells, provides a useful platform to study the development of neutralizing antibodies post-vaccination. Vaccination with the 3 available vaccines all elicited neutralizing serum antibodies that inhibited binding of each of the variant spike proteins to both AT-2 and HLC cells. This study suggests that inhibition of spike binding to target cells may be a more useful technique to assess immunity than gross quantitation of antibody.
Language	English
Publishing date	2023-12-05
Publishing country	New Zealand
Document type	Journal Article
ZDB-ID	2520732-5
ISSN	1179-1535
ISSN	1179-1535
DOI	10.2147/HMER.S431327
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Article ; Online: Suppressive effects of obesity on NK cells: is it time to incorporate obesity as a clinical variable for NK cell-based cancer immunotherapy regimens?

Canter, Robert J / Judge, Sean J / Collins, Craig P / Yoon, Daniel Jaeho / Murphy, William J

Journal for immunotherapy of cancer

2024 Volume 12, Issue 3

MeSH term(s)	Humans ; Killer Cells, Natural ; Immunotherapy ; Neoplasms/therapy ; Obesity/therapy
Language	English
Publishing date	2024-03-13
Publishing country	England
Document type	Journal Article
ZDB-ID	2719863-7
ISSN	2051-1426 ; 2051-1426
ISSN (online)	2051-1426
ISSN	2051-1426
DOI	10.1136/jitc-2023-008443
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Article ; Online: Analysis of SARS-CoV-2 Variant-Specific Serum Antibodies Post-Vaccination Utilizing Immortalized Human Hepatocyte-like Cells (HLC) to Assess Development of Protective Immunity

Collins, Daniel P / Steer, Clifford J

medRxiv

Abstract: Abstract Background: Our previous studies demonstrated that SARS-CoV-2 spike proteins could bind to hepatocytes via the asialoglycoprotein receptor-1 (ASGR-1) facilitating direct infection by the SARS-CoV-2 virus. Immortalized E12-HLC expressed the ... ...

Abstract	Abstract Background: Our previous studies demonstrated that SARS-CoV-2 spike proteins could bind to hepatocytes via the asialoglycoprotein receptor-1 (ASGR-1) facilitating direct infection by the SARS-CoV-2 virus. Immortalized E12-HLC expressed the phenotypic and biological properties of primary human hepatocytes, including their ability to bind spike proteins via ASGR-1 with exception of the spike 1 protein. This binding could be inhibited by spike protein-specific monoclonal antibodies. We used the same spike-blocking analysis to determine if post-vaccination serum was capable of blocking spike protein binding to HLC. Samples collected from subjects prior to, and post-vaccination were quantified for anti-variant-specific antibody (original wild type, alpha (α), beta (β), gamma (γ), and delta (δ) variants by a flow cytometry based immunofluorescent assay. Inhibition of variant spike protein binding to HLC and AT-2 (as a known model for spike 1 binding to the ACE-2 receptor) was analyzed by confocal microscopy. This study was designed to investigate the ability of post-vaccination antibodies to mediate immunity to spike S2, and to validate the utility of the E12-HLC in analyzing that immunity. Methods: Serum was collected from 10 individuals pre- and post-vaccination with the J&J, Moderna or Pfizer vaccines. The serum samples were quantified for variant-specific antibodies in a flow cytometry-based immunofluorescent assay utilizing beads coated with biotinylated variant spike proteins (α, β, γ, δ).Presence of variant-specific antibodies was visualized by anti-human IgG-Alexa 488. Inhibition of spike protein binding to cells was analyzed by immunofluorescent confocal analysis. Biotinylated variant spike proteins were preincubated with serum samples and then tested for binding to target cells. Binding was visualized by Streptavidin-Alexa 594. Results were compared to binding of unblocked spike variants. Results: All variant spike proteins tested bound to both the HLC and AT-2 cells. Pre-vaccination serum samples had no detectable reactivity to any of the variant spike proteins and were unable to inhibit binding of the variant spike proteins to either target cell. Post-vaccination serum samples demonstrated a progression of SARS-CoV-2 antibody levels from low early post-vaccination levels to higher levels at 2.5 months after vaccination. Concurrently, serum samples taken at those different timeframes demonstrated that serum obtained from shortly after vaccination were not as effective in blocking spike protein as serum obtained after 2.5 months post-vaccination. Antibody concentrations were not necessarily associated with better blocking of spike protein binding as spike variant-specific serum antibody concentrations varied significantly between subjects and within each subject. It was also demonstrated that vaccination with all the various available vaccines stimulated antibodies that inhibited binding of the available variant spike proteins to both HLC and AT-2 cells. Conclusion: HLC, along with AT-2 cells, provides a useful platform to study the development of protective antibodies that prevent the binding SARS-CoV-2 spike proteins to target cells. It was shown that vaccination with the three available vaccines all elicited serum antibodies that were protective against binding of each of the variant spike proteins to both AT-2 and HLC cells. This study suggests that analysis of immune serum to block spike binding to target cells may be a more useful technique to assess protective immunity than quantitation of gross antibody alone.
Keywords	covid19
Language	English
Publishing date	2023-08-13
Publisher	Cold Spring Harbor Laboratory Press
Document type	Article ; Online
DOI	10.1101/2023.08.08.23293863
Database	COVID19

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Article: Binding of the SARS-CoV-2 Spike Protein to the Asialoglycoprotein Receptor on Human Primary Hepatocytes and Immortalized Hepatocyte-Like Cells by Confocal Analysis.

Collins, Daniel P / Steer, Clifford J

Hepatic medicine : evidence and research

2021 Volume 13, Page(s) 37–44

Abstract: Background: The SARS-CoV-2 virus may have direct or indirect effects on other human organs beyond the respiratory system and including the liver, via binding of the spike protein. This study investigated the potential direct interactions with the liver ... ...

Abstract	Background: The SARS-CoV-2 virus may have direct or indirect effects on other human organs beyond the respiratory system and including the liver, via binding of the spike protein. This study investigated the potential direct interactions with the liver by comparing the binding of SARS-CoV-2 spike proteins to human AT2-like cells, primary human hepatocytes and immortalized hepatocyte-like hybrid cells. Receptors with binding specificity for SARS-CoV-2 spike protein on AT2 cells and hepatocytes were identified. Methods: The specific binding of biotinylated spike and spike 1 proteins to undifferentiated human E12 MLPC (E12), E12 differentiated alveolar type 2 (AT2) cells, primary human hepatocytes (PHH) and E12 human hepatocyte-like hybrid cells (HLC) was studied by confocal microscopy. We investigated the expression of ACE-2, binding of biotinylated spike protein, biotinylated spike 1 and inhibition of binding by unlabeled spike protein, two neutralizing antibodies and an antibody directed against the hepatocyte asialoglycoprotein receptor 1 (ASGr1). Results: E12 MLPC did not express ACE-2 and did not bind either of spike or spike 1 proteins. AT2-like cells expressed ACE-2 and bound both spike and spike 1. Both PHH and HLC did not express ACE-2 and did not bind spike 1 protein. However, both PHH and HLC actively bound the spike protein. Biotinylated spike protein binding was inhibited by unlabeled spike but not spike 1 protein on PHH and HLC. Two commercial neutralizing antibodies blocked the binding of the spike to PHH and HLC but only one blocked binding to AT2. An antibody to the hepatocyte ASGr1 blocked the binding of the spike protein to PHH and HLC. Conclusion: The absence of ACE-2 receptors and inhibition of spike binding by an antibody to the ASGr1 on both PHH and HLC suggested that the spike protein interacts with the ASGr1. The differential antibody blocking of spike binding to AT2, PHH and HLC indicated that neutralizing activity of SARS-CoV-2 binding might involve additional mechanisms beyond RBD binding to ACE-2.
Language	English
Publishing date	2021-04-14
Publishing country	New Zealand
Document type	Journal Article
ZDB-ID	2520732-5
ISSN	1179-1535
ISSN	1179-1535
DOI	10.2147/HMER.S301979
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Article ; Online: Three-dimensional domain identification in a single hexagonal manganite nanocrystal.

Mokhtar, Ahmed H / Serban, David / Porter, Daniel G / Lichtenberg, Frank / Collins, Stephen P / Bombardi, Alessandro / Spaldin, Nicola A / Newton, Marcus C

Nature communications

2024 Volume 15, Issue 1, Page(s) 3587

Abstract: The three-dimensional domain structure of ferroelectric materials significantly influences their properties. The ferroelectric domain structure of improper multiferroics, such as ... ...

Abstract	The three-dimensional domain structure of ferroelectric materials significantly influences their properties. The ferroelectric domain structure of improper multiferroics, such as YMnO
Language	English
Publishing date	2024-04-27
Publishing country	England
Document type	Journal Article
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-024-48002-z
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Flood-irrigated agriculture mediates climate-induced wetland scarcity for summering sandhill cranes in western North America.

Donnelly, J Patrick / Collins, Daniel P / Knetter, Jeffrey M / Gammonley, James H / Boggie, Matthew A / Grisham, Blake A / Nowak, M Cathy / Naugle, David E

Ecology and evolution

2024 Volume 14, Issue 3, Page(s) e10998

Abstract: Information about species distributions is lacking in many regions of the world, forcing resource managers to answer complex ecological questions with incomplete data. Information gaps are compounded by climate change, driving ecological bottlenecks that ...

Abstract	Information about species distributions is lacking in many regions of the world, forcing resource managers to answer complex ecological questions with incomplete data. Information gaps are compounded by climate change, driving ecological bottlenecks that can act as new demographic constraints on fauna. Here, we construct greater sandhill crane (
Language	English
Publishing date	2024-03-05
Publishing country	England
Document type	Journal Article
ZDB-ID	2635675-2
ISSN	2045-7758
ISSN	2045-7758
DOI	10.1002/ece3.10998
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Article: Metal-Mediated Catalytic Polarization Transfer from

Tickner, Ben J / Dennington, Marcus / Collins, Benjamin G / Gater, Callum A / Tanner, Theo F N / Whitwood, Adrian C / Rayner, Peter J / Watts, Daniel P / Duckett, Simon B

ACS catalysis

2024 Volume 14, Issue 2, Page(s) 994–1004

Abstract: The neutral catalysts [IrCl(H) ...

Abstract	The neutral catalysts [IrCl(H)
Language	English
Publishing date	2024-01-05
Publishing country	United States
Document type	Journal Article
ISSN	2155-5435
ISSN	2155-5435
DOI	10.1021/acscatal.3c05378
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Article ; Online: The Role of Disability Benefits as an Environmental Factor Contributing to Negative Symptoms.

Collins, Delaney E / Luther, Lauren / Raugh, Ian M / Condray, Ruth / Allen, Daniel N / Strauss, Gregory P

Schizophrenia bulletin

2022 Volume 49, Issue 1, Page(s) 1–4

MeSH term(s)	Humans ; Disabled Persons ; Employment ; Disability Evaluation
Language	English
Publishing date	2022-07-08
Publishing country	United States
Document type	Research Support, N.I.H., Extramural ; Editorial ; Research Support, Non-U.S. Gov't
ZDB-ID	439173-1
ISSN	1745-1701 ; 0586-7614
ISSN (online)	1745-1701
ISSN	0586-7614
DOI	10.1093/schbul/sbac077
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Article ; Online: Differentiation of immortalized human multi-lineage progenitor to alveolar type 2-like cells: angiotensin-converting enzyme 2 expression and binding of severe acute respiratory syndrome coronavirus 2 spike and spike 1 proteins.

Collins, Daniel P / Osborn, Mark J / Steer, Clifford J

Cytotherapy

2021 Volume 23, Issue 12, Page(s) 1064–1073

Abstract: Along with the nasal epithelium, the lung epithelium is a portal of entry for sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and many other respiratory viruses. In the case of SARS-CoV-2, the virus surface spike proteins bind to the ... ...

Abstract	Along with the nasal epithelium, the lung epithelium is a portal of entry for sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and many other respiratory viruses. In the case of SARS-CoV-2, the virus surface spike proteins bind to the angiotensin-converting enzyme 2 (ACE-2) receptor to facilitate entry into the respiratory epithelium. Alveolar type 2 (AT2) cells are committed respiratory progenitor cells responsible for the integrity and regeneration of the respiratory epithelium and production of respiratory surfactant proteins. AT2 cells express high levels of surface ACE-2 and thus are a leading target for primary infection by SARS-CoV-2. This study describes a method for directly differentiating telomerase reverse transcriptase-immortalized human cord blood-derived multi-lineage progenitor cells (MLPCs) to AT2-like cells for the purpose of generating an in vitro cellular platform for viral studies. Differentiation was confirmed with the acquisition of AT2 and absence of alveolar type 1 (AT1) specific markers by confocal microscopy. Expression of the ACE-2 receptor was confirmed by immunofluorescence antibody staining, quantitative reverse transcription polymerase chain reaction and binding of biotinylated SARS-CoV-2 spike and spike 1 proteins. The binding of biotinylated spike proteins was specifically blocked by unlabeled spike proteins and neutralizing antibodies. Additionally, it was demonstrated that the spike protein was internalized after binding to the surface membrane of the cells. The authors defined the culture conditions that enabled AT2-like cells to be repeatedly passaged and cryopreserved without further differentiation to AT1. The authors' method provides a stable and renewable source of AT2 cells for respiratory viral binding, blocking and uptake studies.
MeSH term(s)	Angiotensin-Converting Enzyme 2 ; COVID-19 ; Cell Differentiation ; Humans ; SARS-CoV-2 ; Spike Glycoprotein, Coronavirus/genetics
Chemical Substances	Spike Glycoprotein, Coronavirus ; Angiotensin-Converting Enzyme 2 (EC 3.4.17.23)
Language	English
Publishing date	2021-08-17
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2039821-9
ISSN	1477-2566 ; 1465-3249
ISSN (online)	1477-2566
ISSN	1465-3249
DOI	10.1016/j.jcyt.2021.07.017
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Article ; Online: Quantitative mechanical stimulation of GPR68 using a novel 96 well flow plugin.

Segeritz, Philipp / Kolesnik, Kirill / Scott, Daniel J / Collins, David J

Lab on a chip

2024 Volume 24, Issue 6, Page(s) 1616–1625

Abstract: Mechanosensitive proteins play a crucial role in a range of physiological processes, including hearing, tactile sensation and regulating blood flow. While previous work has demonstrated the mechanosensitivity of several proteins, the ability to apply ... ...

Abstract	Mechanosensitive proteins play a crucial role in a range of physiological processes, including hearing, tactile sensation and regulating blood flow. While previous work has demonstrated the mechanosensitivity of several proteins, the ability to apply precisely defined mechanical forces to cells in a consistent, replicable manner remains a significant challenge. In this work we present a novel 96-well plate-compatible plugin device for generating highly-controlled flow-based mechanical simulation of cells, which enables quantitative assessment of mechanosensitive protein function. The device is used to mechanically stimulate HEK 293T cells expressing the mechanosensitive protein GPR68, a G protein-coupled receptor. By assaying intracellular calcium levels during flow-based cell stimulation, we determine that GPR68 signalling is a function of the applied shear-force. As this approach is compatible with conventional cell culture plates and allows for simultaneous readout in a conventional fluorescence plate reader, this represents a valuable new tool to investigate mechanotransduction.
MeSH term(s)	Mechanotransduction, Cellular/physiology ; Cell Culture Techniques ; Receptors, G-Protein-Coupled/metabolism ; Stress, Mechanical
Chemical Substances	Receptors, G-Protein-Coupled
Language	English
Publishing date	2024-03-12
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2056646-3
ISSN	1473-0189 ; 1473-0197
ISSN (online)	1473-0189
ISSN	1473-0197
DOI	10.1039/d3lc00767g
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