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Updated listing of COVID-19 vaccine and therapeutic trials from NIH Clinical Trials.gov

Curator: Stephen J. Williams, PhD

 

The following file contains an updated list (search on 4/15/2020) of COVID-19 related clinical trials from https://clinicaltrials.gov/

 

The Excel file can be uploaded here: Current Covid-19 Trials

 

Each sheet in the workbook is separated by current COVID-19 vaccine trials, currents COVID-19 trials with the IL6R (interleukin 6 receptor) antagonist tocilizumab, and all COVID related trials.  The Excel spreadsheet also contains links to more information about the trials.

 

As of April 15, 2020 the number of listed trials are as follows:

 

clinicaltrials.gov search terms Number of results Number of completed  trials Number of trials currently recruiting
COVID-19 or SARS-CoV-2 410 5 completed

5 withdrawn  

192
1st row terms + vaccine 28 0 15
1st row terms + tocilizumab 16 0 10
1st row terms + hydroxychloroquine 61 1 22

 

A few highlights of the COVID related trials on clinicaltrials.gov

 

Withdrawn trials

 

Recombinant Human Angiotensin-converting Enzyme 2 (rhACE2) as a Treatment for Patients With COVID-19 (NCT04287686)

Study Description

Go to 

Brief Summary:

This is an open label, randomized, controlled, pilot clinical study in patients with COVID-19, to obtain preliminary biologic, physiologic, and clinical data in patients with COVID-19 treated with rhACE2 or control patients, to help determine whether a subsequent Phase 2B trial is warranted.

 

Condition or disease  Intervention/treatment  Phase 
COVID-19 Drug: Recombinant human angiotensin-converting enzyme 2 (rhACE2) Not Applicable

 

Detailed Description:

This is a small pilot study investigating whether there is any efficacy signal that warrants a larger Phase 2B trial, or any harm that suggests that such a trial should not be done. It is not expected to produce statistically significant results in the major endpoints. The investigators will examine all of the biologic, physiological, and clinical data to determine whether a Phase 2B trial is warranted.

Primary efficacy analysis will be carried only on patients receiving at least 4 doses of active drug. Safety analysis will be carried out on all patients receiving at least one dose of active drug.

It is planned to enroll more than or equal to 24 subjects with COVID-19. It is expected to have at least 12 evaluable patients in each group.

Experimental group: 0.4 mg/kg rhACE2 IV BID and standard of care Control group: standard of care

Intervention duration: up to 7 days of therapy

No planned interim analysis.

Study was withdrawn before participants were enrolled.

Washed Microbiota Transplantation for Patients With 2019-nCoV Infection (NCT04251767)

Study Description

Go to 

Brief Summary:

Gut dysbiosis co-exists in patients with coronavirus pneumonia. Some of these patients would develop secondary bacterial infections and antibiotic-associated diarrhea (AAD). The recent study on using washed microbiota transplantation (WMT) as rescue therapy in critically ill patients with AAD demonstrated the important clinical benefits and safety of WMT. This clinical trial aims to evaluate the outcome of WMT combining with standard therapy for patients with 2019-novel coronavirus pneumonia, especially for those patients with dysbiosis-related conditions.

 

Detailed Description:

An ongoing outbreak of 2019 novel coronavirus was reported in Wuhan, China. 2019-nCoV has caused a cluster of pneumonia cases, and posed continuing epidemic threat to China and even global health. Unfortunately, there is currently no specific effective treatment for the viral infection and the related serious complications. It is in urgent need to find a new specific effective treatment for the 2019-nCoV infection. According to Declaration of Helsinki and International Ethical Guidelines for Health-related Research Involving Humans, the desperately ill patients with 2019-nCov infection during disease outbreaks have a moral right to try unvalidated medical interventions (UMIs) and that it is therefore unethical to restrict access to UMIs to the clinical trial context.

There is a vital link between the intestinal tract and respiratory tract, which was exemplified by intestinal complications during respiratory disease and vice versa. Some of these patients can develop secondary bacterial infections and antibiotic-associated diarrhea (AAD). The recent study on using washed microbiota transplantation (WMT) as rescue therapy in critically ill patients with AAD demonstrated the important clinical benefits and safety of WMT. Additionally, the recent animal study provided direct evidence supporting that antibiotics could decrease gut microbiota and the lung stromal interferon signature and facilitate early influenza virus replication in lung epithelia. Importantly, the above antibiotics caused negative effects can be reversed by fecal microbiota transplantation (FMT) which suggested that FMT might be able to induce a significant improvement in the respiratory virus infection. Another evidence is that the microbiota could confer protection against certain virus infection such as influenza virus and respiratory syncytial virus by priming the immune response to viral evasion. The above results suggested that FMT might be a new therapeutic option for the treatment of virus-related pneumonia. The methodology of FMT recently was coined as WMT, which is dependent on the automatic facilities and washing process in a laboratory room. Patients underwent WMT with the decreased rate of adverse events and unchanged clinical efficacy in ulcerative colitis and Crohn’s disease. This clinical trial aims to evaluate the outcome of WMT combining with standard therapy for patients with novel coronavirus pneumonia, especially for those patients with dysbiosis-related conditions.

 

Responsible Party: Faming Zhang, Director of Medical Center for Digestive Diseases, The Second Hospital of Nanjing Medical University
Identifier NCT04251767     History of Changes

Study was withdrawn before participants were enrolled.

 

Therapy for Pneumonia Patients iInfected by 2019 Novel Coronavirus (NCT04293692)

Study Description

Go to 

Brief Summary:

The 2019 novel coronavirus pneumonia outbroken in Wuhan, China, which spread quickly to 26 countries worldwide and presented a serious threat to public health. It is mainly characterized by fever, dry cough, shortness of breath and breathing difficulties. Some patients may develop into rapid and deadly respiratory system injury with overwhelming inflammation in the lung. Currently, there is no effective treatment in clinical practice. The present clinical trial is to explore the safety and efficacy of Human Umbilical Cord Mesenchymal Stem Cells (UC-MSCs) therapy for novel coronavirus pneumonia patients.

Detailed Description:

Since late December 2019, human pneumonia cases infected by a novel coronavirus (2019-nCoV) were firstly identified in Wuhan, China. As the virus is contagious and of great epidemic, more and more cases have found in other areas of China and abroad. Up to February 24, a total of 77, 779 confirmed cases were reported in China. At present, there is no effective treatment for patients identified with novel coronavirus pneumonia. Therefore, it’s urgent to explore more active therapeutic methods to cure the patients.

Recently, some clinical researches about the 2019 novel coronavirus pneumonia published in The Lancet and The New England Journal of Medicine suggested that massive inflammatory cell infiltration and inflammatory cytokines secretion were found in patients’ lungs, alveolar epithelial cells and capillary endothelial cells were damaged, causing acute lung injury. It seems that the key to cure the pneumonia is to inhibit the inflammatory response, resulting to reduce the damage of alveolar epithelial cells and endothelial cells and repair the function of the lung.

Mesenchymal stem cells (MSCs) are widely used in basic research and clinical application. They are proved to migrate to damaged tissues, exert anti-inflammatory and immunoregulatory functions, promote the regeneration of damaged tissues and inhibit tissue fibrosis. Studies have shown that MSCs can significantly reduce acute lung injury in mice caused by H9N2 and H5N1 viruses by reducing the levels of proinflammatory cytokines and the recruitment of inflammatory cells into the lungs. Compared with MSCs from other sources, human umbilical cord-derived MSCs (UC-MSCs) have been widely applied to various diseases due to their convenient collection, no ethical controversy, low immunogenicity, and rapid proliferation rate. In our recent research, we confirmed that UC-MSCs can significantly reduce inflammatory cell infiltration and inflammatory factors expression in lung tissue, and significantly protect lung tissue from endotoxin (LPS) -induced acute lung injury in mice.

The purpose of this clinical study is to investigate safety and efficiency of UC-MSCs in treating pneumonia patients infected by 2019-nCoV. The investigators planned to recruit 48 patients aged from 18 to 75 years old and had no severe underlying diseases. In the cell treatment group, 24 patients received 0.5*10E6 UC-MSCs /kg body weight intravenously treatment 4 times every other day besides conventional treatment. In the control group, other 24 patients received conventional treatment plus 4 times of placebo intravenously. The lung CT, blood biochemical examination, lymphocyte subsets, inflammatory factors, 28-days mortality, etc will be evaluated within 24h and 1, 2, 4, 8 weeks after UC-MSCs treatment.

Sponsor:

Puren Hospital Affiliated to Wuhan University of Science and Technology

Collaborator:

Wuhan Hamilton Bio-technology Co., Ltd

Study was withdrawn before participants were enrolled.

 

Prognositc Factors in COVID-19 Patients Complicated With Hypertension (NCT04272710)

Study Description

Brief Summary:

There are currently no clinical studies reporting clinical characteristics difference between the hypertension patients with and without ACEI treatment when suffered with novel coronavirus infection in China

Detailed Description:

At present, the outbreak of the new coronavirus (2019-nCoV) infection in Wuhan and Hubei provinces has attracted great attention from the medical community across the country. Both 2019-nCoV and SARS viruses are coronaviruses, and they have a large homology.

Published laboratory studies have suggested that SARS virus infection and its lung injury are related to angiotensin-converting enzyme 2 (ACE2) in lung tissue. And ACE and ACE2 in the renin-angiotensin system (RAS) are vital central links to maintain hemodynamic stability and normal heart and kidney function in vivo.

A large amount of evidence-based medical evidence shows that ACE inhibitors are the basic therapeutic drugs for maintaining hypertension, reducing the risk of cardiovascular, cerebrovascular, and renal adverse events, improving quality of life, and prolonging life in patients with hypertension. Recent experimental studies suggest that treatment with ACE inhibitors can significantly reduce pulmonary inflammation and cytokine release caused by coronavirus infection.

 

ACEI treatment

hypertension patients with ACEI treatment when suffered with novel coronavirus infection in China

Control

hypertension patients without ACEI treatment when suffered with novel coronavirus infection in China

 

Locations

China
The First Affiliated Hospital of Chongqing Medical University Chongqing, China

Sponsors and Collaborators Chongqing Medical University

 

Responsible PI: Dongying Zhang, Associate Professor, Chongqing Medical University

Withdrawn (Similar projects have been registered, and it needs to be withdrawn.)

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Structure-guided Drug Discovery: (1) The Coronavirus 3CL hydrolase (Mpro) enzyme (main protease) essential for proteolytic maturation of the virus and (2) viral protease, the RNA polymerase, the viral spike protein, a viral RNA as promising two targets for discovery of cleavage inhibitors of the viral spike polyprotein preventing the Coronavirus Virion the spread of infection

 

Curators and Reporters: Stephen J. Williams, PhD and Aviva Lev-Ari, PhD, RN

 

Therapeutical options to coronavirus (2019-nCoV) include consideration of the following:

(a) Monoclonal and polyclonal antibodies

(b)  Vaccines

(c)  Small molecule treatments (e.g., chloroquinolone and derivatives), including compounds already approved for other indications 

(d)  Immuno-therapies derived from human or other sources

 

 

Structure of the nCoV trimeric spike

The World Health Organization has declared the outbreak of a novel coronavirus (2019-nCoV) to be a public health emergency of international concern. The virus binds to host cells through its trimeric spike glycoprotein, making this protein a key target for potential therapies and diagnostics. Wrapp et al. determined a 3.5-angstrom-resolution structure of the 2019-nCoV trimeric spike protein by cryo–electron microscopy. Using biophysical assays, the authors show that this protein binds at least 10 times more tightly than the corresponding spike protein of severe acute respiratory syndrome (SARS)–CoV to their common host cell receptor. They also tested three antibodies known to bind to the SARS-CoV spike protein but did not detect binding to the 2019-nCoV spike protein. These studies provide valuable information to guide the development of medical counter-measures for 2019-nCoV. [Bold Face Added by ALA]

Science, this issue p. 1260

Abstract

The outbreak of a novel coronavirus (2019-nCoV) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure development, we determined a 3.5-angstrom-resolution cryo–electron microscopy structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also provide biophysical and structural evidence that the 2019-nCoV S protein binds angiotensin-converting enzyme 2 (ACE2) with higher affinity than does severe acute respiratory syndrome (SARS)-CoV S. Additionally, we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 2019-nCoV S, suggesting that antibody cross-reactivity may be limited between the two RBDs. The structure of 2019-nCoV S should enable the rapid development and evaluation of medical countermeasures to address the ongoing public health crisis.

SOURCE
Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation
  1. Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA.

  2. 2Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
  1. Corresponding author. Email: jmclellan@austin.utexas.edu
  1. * These authors contributed equally to this work.

Science  13 Mar 2020:
Vol. 367, Issue 6483, pp. 1260-1263
DOI: 10.1126/science.abb2507

 

02/04/2020

New Coronavirus Protease Structure Available

PDB data provide a starting point for structure-guided drug discovery

A high-resolution crystal structure of COVID-19 (2019-nCoV) coronavirus 3CL hydrolase (Mpro) has been determined by Zihe Rao and Haitao Yang’s research team at ShanghaiTech University. Rapid public release of this structure of the main protease of the virus (PDB 6lu7) will enable research on this newly-recognized human pathogen.

Recent emergence of the COVID-19 coronavirus has resulted in a WHO-declared public health emergency of international concern. Research efforts around the world are working towards establishing a greater understanding of this particular virus and developing treatments and vaccines to prevent further spread.

While PDB entry 6lu7 is currently the only public-domain 3D structure from this specific coronavirus, the PDB contains structures of the corresponding enzyme from other coronaviruses. The 2003 outbreak of the closely-related Severe Acute Respiratory Syndrome-related coronavirus (SARS) led to the first 3D structures, and today there are more than 200 PDB structures of SARS proteins. Structural information from these related proteins could be vital in furthering our understanding of coronaviruses and in discovery and development of new treatments and vaccines to contain the current outbreak.

The coronavirus 3CL hydrolase (Mpro) enzyme, also known as the main protease, is essential for proteolytic maturation of the virus. It is thought to be a promising target for discovery of small-molecule drugs that would inhibit cleavage of the viral polyprotein and prevent spread of the infection.

Comparison of the protein sequence of the COVID-19 coronavirus 3CL hydrolase (Mpro) against the PDB archive identified 95 PDB proteins with at least 90% sequence identity. Furthermore, these related protein structures contain approximately 30 distinct small molecule inhibitors, which could guide discovery of new drugs. Of particular significance for drug discovery is the very high amino acid sequence identity (96%) between the COVID-19 coronavirus 3CL hydrolase (Mpro) and the SARS virus main protease (PDB 1q2w). Summary data about these closely-related PDB structures are available (CSV) to help researchers more easily find this information. In addition, the PDB houses 3D structure data for more than 20 unique SARS proteins represented in more than 200 PDB structures, including a second viral protease, the RNA polymerase, the viral spike protein, a viral RNA, and other proteins (CSV).

Public release of the COVID-19 coronavirus 3CL hydrolase (Mpro), at a time when this information can prove most vital and valuable, highlights the importance of open and timely availability of scientific data. The wwPDB strives to ensure that 3D biological structure data remain freely accessible for all, while maintaining as comprehensive and accurate an archive as possible. We hope that this new structure, and those from related viruses, will help researchers and clinicians address the COVID-19 coronavirus global public health emergency.

Update: Released COVID-19-related PDB structures include

  • PDB structure 6lu7 (X. Liu, B. Zhang, Z. Jin, H. Yang, Z. Rao Crystal structure of COVID-19 main protease in complex with an inhibitor N3 doi: 10.2210/pdb6lu7/pdb) Released 2020-02-05
  • PDB structure 6vsb (D. Wrapp, N. Wang, K.S. Corbett, J.A. Goldsmith, C.-L. Hsieh, O. Abiona, B.S. Graham, J.S. McLellan (2020) Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Science doi: 10.1126/science.abb2507) Released 2020-02-26
  • PDB structure 6lxt (Y. Zhu, F. Sun Structure of post fusion core of 2019-nCoV S2 subunit doi: 10.2210/pdb6lxt/pdb) Released 2020-02-26
  • PDB structure 6lvn (Y. Zhu, F. Sun Structure of the 2019-nCoV HR2 Domain doi: 10.2210/pdb6lvn/pdb) Released 2020-02-26
  • PDB structure 6vw1
    J. Shang, G. Ye, K. Shi, Y.S. Wan, H. Aihara, F. Li Structural basis for receptor recognition by the novel coronavirus from Wuhan doi: 10.2210/pdb6vw1/pdb
    Released 2020-03-04
  • PDB structure 6vww
    Y. Kim, R. Jedrzejczak, N. Maltseva, M. Endres, A. Godzik, K. Michalska, A. Joachimiak, Center for Structural Genomics of Infectious Diseases Crystal Structure of NSP15 Endoribonuclease from SARS CoV-2 doi: 10.2210/pdb6vww/pdb
    Released 2020-03-04
  • PDB structure 6y2e
    L. Zhang, X. Sun, R. Hilgenfeld Crystal structure of the free enzyme of the SARS-CoV-2 (2019-nCoV) main protease doi: 10.2210/pdb6y2e/pdb
    Released 2020-03-04
  • PDB structure 6y2f
    L. Zhang, X. Sun, R. Hilgenfeld Crystal structure (monoclinic form) of the complex resulting from the reaction between SARS-CoV-2 (2019-nCoV) main protease and tert-butyl (1-((S)-1-(((S)-4-(benzylamino)-3,4-dioxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-3-cyclopropyl-1-oxopropan-2-yl)-2-oxo-1,2-dihydropyridin-3-yl)carbamate (alpha-ketoamide 13b) doi: 10.2210/pdb6y2f/pdb
    Released 2020-03-04
  • PDB structure 6y2g
    L. Zhang, X. Sun, R. Hilgenfeld Crystal structure (orthorhombic form) of the complex resulting from the reaction between SARS-CoV-2 (2019-nCoV) main protease and tert-butyl (1-((S)-1-(((S)-4-(benzylamino)-3,4-dioxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-3-cyclopropyl-1-oxopropan-2-yl)-2-oxo-1,2-dihydropyridin-3-yl)carbamate (alpha-ketoamide 13b) doi: 10.2210/pdb6y2g/pdb
    Released 2020-03-04
First page image

Abstract

Coronavirus disease 2019 (COVID-19) is a global pandemic impacting nearly 170 countries/regions and more than 285,000 patients worldwide. COVID-19 is caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which invades cells through the angiotensin converting enzyme 2 (ACE2) receptor. Among those with COVID-19, there is a higher prevalence of cardiovascular disease and more than 7% of patients suffer myocardial injury from the infection (22% of the critically ill). Despite ACE2 serving as the portal for infection, the role of ACE inhibitors or angiotensin receptor blockers requires further investigation. COVID-19 poses a challenge for heart transplantation, impacting donor selection, immunosuppression, and post-transplant management. Thankfully there are a number of promising therapies under active investigation to both treat and prevent COVID-19. Key Words: COVID-19; myocardial injury; pandemic; heart transplant

SOURCE

https://www.ahajournals.org/doi/pdf/10.1161/CIRCULATIONAHA.120.046941

ACE2

  • Towler P, Staker B, Prasad SG, Menon S, Tang J, Parsons T, Ryan D, Fisher M, Williams D, Dales NA, Patane MA, Pantoliano MW (Apr 2004). “ACE2 X-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis”The Journal of Biological Chemistry279 (17): 17996–8007. doi:10.1074/jbc.M311191200PMID 14754895.

 

  • Turner AJ, Tipnis SR, Guy JL, Rice G, Hooper NM (Apr 2002). “ACEH/ACE2 is a novel mammalian metallocarboxypeptidase and a homologue of angiotensin-converting enzyme insensitive to ACE inhibitors”Canadian Journal of Physiology and Pharmacology80 (4): 346–53. doi:10.1139/y02-021PMID 12025971.

 

  •  Zhang, Haibo; Penninger, Josef M.; Li, Yimin; Zhong, Nanshan; Slutsky, Arthur S. (3 March 2020). “Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target”Intensive Care Medicine. Springer Science and Business Media LLC. doi:10.1007/s00134-020-05985-9ISSN 0342-4642PMID 32125455.

 

  • ^ Gurwitz, David (2020). “Angiotensin receptor blockers as tentative SARS‐CoV‐2 therapeutics”Drug Development Researchdoi:10.1002/ddr.21656PMID 32129518.

 

Angiotensin converting enzyme 2 (ACE2)

is an exopeptidase that catalyses the conversion of angiotensin I to the nonapeptide angiotensin[1-9][5] or the conversion of angiotensin II to angiotensin 1-7.[6][7] ACE2 has direct effects on cardiac functiona and is expressed predominantly in vascular endothelial cells of the heart and the kidneys.[8] ACE2 is not sensitive to the ACE inhibitor drugs used to treat hypertension.[9]

ACE2 receptors have been shown to be the entry point into human cells for some coronaviruses, including the SARS virus.[10] A number of studies have identified that the entry point is the same for SARS-CoV-2,[11] the virus that causes COVID-19.[12][13][14][15]

Some have suggested that a decrease in ACE2 could be protective against Covid-19 disease[16], but others have suggested the opposite, that Angiotensin II receptor blocker drugs could be protective against Covid-19 disease via increasing ACE2, and that these hypotheses need to be tested by datamining of clinical patient records.[17]

REFERENCES

https://en.wikipedia.org/wiki/Angiotensin-converting_enzyme_2

 

FOLDING@HOME TAKES UP THE FIGHT AGAINST COVID-19 / 2019-NCOV

We need your help! Folding@home is joining researchers around the world working to better understand the 2019 Coronavirus (2019-nCoV) to accelerate the open science effort to develop new life-saving therapies. By downloading Folding@Home, you can donate your unused computational resources to the Folding@home Consortium, where researchers working to advance our understanding of the structures of potential drug targets for 2019-nCoV that could aid in the design of new therapies. The data you help us generate will be quickly and openly disseminated as part of an open science collaboration of multiple laboratories around the world, giving researchers new tools that may unlock new opportunities for developing lifesaving drugs.

2019-nCoV is a close cousin to SARS coronavirus (SARS-CoV), and acts in a similar way. For both coronaviruses, the first step of infection occurs in the lungs, when a protein on the surface  of the virus binds to a receptor protein on a lung cell. This viral protein is called the spike protein, depicted in red in the image below, and the receptor is known as ACE2. A therapeutic antibody is a type of protein that can block the viral protein from binding to its receptor, therefore preventing the virus from infecting the lung cell. A therapeutic antibody has already been developed for SARS-CoV, but to develop therapeutic antibodies or small molecules for 2019-nCoV, scientists need to better understand the structure of the viral spike protein and how it binds to the human ACE2 receptor required for viral entry into human cells.

Proteins are not stagnant—they wiggle and fold and unfold to take on numerous shapes.  We need to study not only one shape of the viral spike protein, but all the ways the protein wiggles and folds into alternative shapes in order to best understand how it interacts with the ACE2 receptor, so that an antibody can be designed. Low-resolution structures of the SARS-CoV spike protein exist and we know the mutations that differ between SARS-CoV and 2019-nCoV.  Given this information, we are uniquely positioned to help model the structure of the 2019-nCoV spike protein and identify sites that can be targeted by a therapeutic antibody. We can build computational models that accomplish this goal, but it takes a lot of computing power.

This is where you come in! With many computers working towards the same goal, we aim to help develop a therapeutic remedy as quickly as possible. By downloading Folding@home here [LINK] and selecting to contribute to “Any Disease”, you can help provide us with the computational power required to tackle this problem. One protein from 2019-nCoV, a protease encoded by the viral RNA, has already been crystallized. Although the 2019-nCoV spike protein of interest has not yet been resolved bound to ACE2, our objective is to use the homologous structure of the SARS-CoV spike protein to identify therapeutic antibody targets.

This illustration, created at the Centers for Disease Control and Prevention (CDC), reveals ultrastructural morphology exhibited by coronaviruses. Note the spikes that adorn the outer surface of the virus, which impart the look of a corona surrounding the virion, when viewed electron microscopically. A novel coronavirus virus was identified as the cause of an outbreak of respiratory illness first detected in Wuhan, China in 2019.

Image and Caption Credit: Alissa Eckert, MS; Dan Higgins, MAM available at https://phil.cdc.gov/Details.aspx?pid=23311

Structures of the closely related SARS-CoV spike protein bound by therapeutic antibodies may help rapidly design better therapies. The three monomers of the SARS-CoV spike protein are shown in different shades of red; the antibody is depicted in green. [PDB: 6NB7 https://www.rcsb.org/structure/6nb7]

(post authored by Ariana Brenner Clerkin)

References:

PDB 6lu7 structure summary ‹ Protein Data Bank in Europe (PDBe) ‹ EMBL-EBI https://www.ebi.ac.uk/pdbe/entry/pdb/6lu7 (accessed Feb 5, 2020).

Tian, X.; Li, C.; Huang, A.; Xia, S.; Lu, S.; Shi, Z.; Lu, L.; Jiang, S.; Yang, Z.; Wu, Y.; et al. Potent Binding of 2019 Novel Coronavirus Spike Protein by a SARS Coronavirus-Specific Human Monoclonal Antibody; preprint; Microbiology, 2020. https://doi.org/10.1101/2020.01.28.923011.

Walls, A. C.; Xiong, X.; Park, Y. J.; Tortorici, M. A.; Snijder, J.; Quispe, J.; Cameroni, E.; Gopal, R.; Dai, M.; Lanzavecchia, A.; et al. Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion. Cell 2019176, 1026-1039.e15. https://doi.org/10.2210/pdb6nb7/pdb.

SOURCE

https://foldingathome.org/2020/02/27/foldinghome-takes-up-the-fight-against-covid-19-2019-ncov/

UPDATED 3/13/2020

I am reposting the following Science blog post from Derrick Lowe as is and ask people go browse through the comments on his Science blog In the Pipeline because, as Dr. Lowe states that in this current crisis it is important to disseminate good information as quickly as possible so wanted the readers here to have the ability to read his great posting on this matter of Covid-19.  Also i would like to direct readers to the journal Science opinion letter concerning how important it is to rebuild the trust in good science and the scientific process.  The full link for the following In the Pipeline post is: https://blogs.sciencemag.org/pipeline/archives/2020/03/06/covid-19-small-molecule-therapies-reviewed

A Summary of current potential repurposed therapeutics for COVID-19 Infection from In The Pipeline: A Science blog from Derick Lowe

Covid-19 Small Molecule Therapies Reviewed

Let’s take inventory on the therapies that are being developed for the coronavirus epidemic. Here is a very thorough list of at Biocentury, and I should note that (like Stat and several other organizations) they’re making all their Covid-19 content free to all readers during this crisis. I’d like to zoom in today on the potential small-molecule therapies, since some of these have the most immediate prospects for use in the real world.

The ones at the front of the line are repurposed drugs that are already approved for human use, for a lot of obvious reasons. The Biocentury list doesn’t cover these, but here’s an article at Nature Biotechnology that goes into detail. Clinical trials are a huge time sink – they sort of have to be, in most cases, if they’re going to be any good – and if you’ve already done all that stuff it’s a huge leg up, even if the drug itself is not exactly a perfect fit for the disease. So what do we have? The compound that is most advanced is probably remdesivir from Gilead, at right. This has been in development for a few years as an RNA virus therapy – it was originally developed for Ebola, and has been tried out against a whole list of single-strand RNA viruses. That includes the related coronaviruses SARS and MERS, so Covid-19 was an obvious fit.

The compound is a prodrug – that phosphoramide gets cleaved off completely, leaving the active 5-OH compound GS-44-1524. It mechanism of action is to get incorporated into viral RNA, since it’s taken up by RNA polymerase and it largely seems to evade proofreading. This causes RNA termination trouble later on, since that alpha-nitrile C-nucleoside is not exactly what the virus is expecting in its genome at that point, and thus viral replication is inhibited.

There are five clinical trials underway (here’s an overview at Biocentury). The NIH has an adaptive-design Phase II trial that has already started in Nebraska, with doses to be changed according to Bayesian readouts along the way. There are two Phase III trials underway at China-Japan Friendship Hospital in Hubei, double-blinded and placebo-controlled (since placebo is, as far as drug therapy goes, the current standard of care). And Gilead themselves are starting two open-label trials, one with no control arm and one with an (unblinded) standard-of-care comparison arm. Those might read out first, depending on when they get off the ground, but will be only rough readouts due to the fast-and-loose trial design. The two Hubei trials and the NIH one will add some rigor to the process, but I’m not sure when they’re going to report. My personal opinion is that I like the chances of this drug more than anything else on this list, but it’s still unlikely to be a game-changer.

There’s an RNA polymerase inhibitor (favipiravir) from Toyama, at right, that’s in a trial in China. It’s a thought – a broad-spectrum agent of this sort would be the sort of thing to try. But unfortunately, from what I can see, it has already turned up as ineffective in in vitro tests. The human trial that’s underway is honestly the sort of thing that would only happen under circumstances like the present: a developing epidemic with a new pathogen and no real standard of care. I hold out little hope for this one, but given that there’s nothing else at present, it probably should be tried. As you’ll see, this is far from the only situation like this.

One of the screens of known drugs in China that also flagged remdesivir noted that the old antimalarial drug chloroquine seemed to be effective in vitro. It had been reported some years back as a possible antiviral, working through more than one mechanism, probably both at viral entry and intracellularly thereafter. That part shouldn’t be surprising – chloroquine’s actual mode(s) of action against malaria parasites are still not completely worked out, either, and some of what people thought they knew about it has turned out to be wrong. There are several trials underway with it at Chinese facilities, some in combination with other agents like remdesivir. Chloroquine has of course been taken for many decades as an antimalarial, but it has a number of liabilities, including seizures, hearing damage, retinopathy and sudden effects on blood glucose. So it’s going to be important to establish just how effective it is and what doses will be needed. Just as with vaccine candidates, it’s possible to do more harm with a rushed treatment than the disease is doing itself

There are several other known antiviral drugs are being tried in China, but I don’t have too much hope for those, either. The neuraminidase inhibitors such as oseltamivir (better known as Tamiflu) were tried against SARS and were ineffective; there is no reason to expect anything versus Covid-19 although these drugs are a component of some drug cocktail trials. The HIV protease therapies such as darunavir and the combination therapy Kaletra are in trials, but that’s also a rather desperate long shot, since there’s no particular reason to think that they will have any such protease inhibition against what this new virus has to offer (and indeed, such agents weren’t much help against SARS in the end, either). The classic interferon/ribavirin combination seems to have had some activity against SARS and MERS, and is in two trials from what I can see. That’s not an awful idea by any means, but it’s not a great one, either: if your viral disease has interferon/ribavirin as a front line therapy, it generally means that there’s nothing really good available. No, unless we get really lucky none of these ideas are going to slow the disease down much.

There are a few other repurposed-protease-inhibitors ideas out there, such as this one. (Edit: I had seen this paper but couldn’t track it down, so thanks to those who sent it along). This paper suggests that the TMPRSS2 protease is important for viral entry on the human-cell-side of the process, a pathway that has been noted for other coronaviruses. And it points out that there is a an approved inhibitor (in Japan) for this enzyme (camostat), so that would definitely seem to be worth a trial, probably in combination with remdesivir.

That’s about it for the existing small molecules, from what I can see. What about new ones? Don’t hold your breath, is all I can say. A drug discovery program from scratch against a new pathogen is, as many readers here well know, not a trivial exercise. As this Bloomberg article details, many such efforts in the past (small molecules and vaccines alike) have come to grief because by the time they had anything to deliver the epidemic itself had passed. Indeed, Gilead’s remdesivir had already been dropped as a potential Ebola therapy.

You will either need to have a target in mind up front or go phenotypic. For the former, what you’d see are better characterizations of the viral protease and more extensive screens against it. Two other big target areas are viral entry (which involves the “spike” proteins on the virus surface and the ACE2 protein on human cells) and viral replication. To the former, it’s worth quickly noting that ACE2 is so much unlike the more familiar ACE protein that none of the cardiovascular ACE inhibitors do anything to it at all. And targeting the latter mechanisms is how remdesivir was developed as a possible Ebola agent, but as you can see, that took time, too. Phenotypic screens are perfectly reasonable against viral pathogens as well, but you’ll need to put time and effort into that assay up front, just as with any phenotypic effort, because as anyone who does that sort of work will tell you, a bad phenotypic screen is a complete waste of everyone’s time.

One of the key steps for either route is identifying an animal model. While animal models of infectious disease can be extremely well translated to human therapy, that doesn’t happen by accident: you need to choose the right animal. Viruses in general (and coronaviruses are no exception) vary widely in their effects in different species, and not just across the gaps of bird/reptile/human and the like. No, you’ll run into things where even the usual set of small mammals are acting differently from each other, with some of them not even getting sick at all. This current virus may well have gone through a couple of other mammalian species before landing on us, but you’ll note that dogs (to pick one) don’t seem to have any problem with it.

All this means that any new-target new-chemical-matter effort against Covid-19 (or any new pathogen) is going to take years, and there is just no way around that. Update: see here for just such an effort to start finding fragment hits for the viral protease. This puts small molecules in a very bimodal distribution: you have the existing drugs that might be repurposed, and are presumably available right now. Nothing else is! At the other end, for completely new therapies you have the usual prospects of drug discovery: years from now, lots of money, low success rate, good luck to all of us. The gap between these two could in theory be filled by vaccines and antibody therapies (if everything goes really, really well) but those are very much their own area and will be dealt with in a separate post.

Either way, the odds are that we (and I mean “we as a species” here) are going to be fighting this epidemic without any particularly amazing pharmacological weapons. Eventually we’ll have some, but I would advise people, pundits, and politicians not to get all excited about the prospects for some new therapies to come riding up over the hill to help us out. The odds of that happening in time to do anything about the current outbreak are very small. We will be going for months, years, with the therapeutic options we have right now. Look around you: what we have today is what we have to work with.

Other related articles published in this Open Access Online Scientific Journal include the following:

 

Group of Researchers @ University of California, Riverside, the University of Chicago, the U.S. Department of Energy’s Argonne National Laboratory, and Northwestern University solve COVID-19 Structure and Map Potential Therapeutics

Reporters: Stephen J Williams, PhD and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2020/03/06/group-of-researchers-solve-covid-19-structure-and-map-potential-therapeutic/

Predicting the Protein Structure of Coronavirus: Inhibition of Nsp15 can slow viral replication and Cryo-EM – Spike protein structure (experimentally verified) vs AI-predicted protein structures (not experimentally verified) of DeepMind (Parent: Google) aka AlphaFold

Curators: Stephen J. Williams, PhD and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2020/03/08/predicting-the-protein-structure-of-coronavirus-inhibition-of-nsp15-can-slow-viral-replication-and-cryo-em-spike-protein-structure-experimentally-verified-vs-ai-predicted-protein-structures-not/

 

Coronavirus facility opens at Rambam Hospital using new Israeli tech

https://www.jpost.com/Israel-News/Coronavirus-facility-opens-at-Rambam-Hospital-using-new-Israeli-tech-619681

 

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

Effective humoral immune responses to infection and immunization are defined by high-affinity antibodies generated as a result of B cell differentiation and selection that occurs within germinal centers (GC). Within the GC, B cells undergo affinity maturation, an iterative and competitive process wherein B cells mutate their immunoglobulin genes (somatic hypermutation) and undergo clonal selection by competing for T cell help. Balancing the decision to remain within the GC and continue participating in affinity maturation or to exit the GC as a plasma cell (PC) or memory B cell (MBC) is critical for achieving optimal antibody avidity, antibody quantity, and establishing immunological memory in response to immunization or infection. Humoral immune responses during chronic infections are often dysregulated and characterized by hypergammaglobulinemia, decreased affinity maturation, and delayed development of neutralizing antibodies. Previous studies have suggested that poor antibody quality is in part due to deletion of B cells prior to establishment of the GC response.

 

In fact the impact of chronic infections on B cell fate decisions in the GC remains poorly understood. To address this question, researchers used single-cell transcriptional profiling of virus-specific GC B cells to test the hypothesis that chronic viral infection disrupted GC B cell fate decisions leading to suboptimal humoral immunity. These studies revealed a critical GC differentiation checkpoint that is disrupted by chronic infection, specifically at the point of dark zone re-entry. During chronic viral infection, virus-specific GC B cells were shunted towards terminal plasma cell (PC) or memory B cell (MBC) fates at the expense of continued participation in the GC. Early GC exit was associated with decreased B cell mutational burden and antibody quality. Persisting antigen and inflammation independently drove facets of dysregulation, with a key role for inflammation in directing premature terminal GC B cell differentiation and GC exit. Thus, the present research defines GC defects during chronic viral infection and identify a critical GC checkpoint that is short-circuited, preventing optimal maturation of humoral immunity.

 

Together, these studies identify a key GC B cell differentiation checkpoint that is dysregulated during chronic infection. Further, it was found that the chronic inflammatory environment, rather than persistent antigen, is sufficient to drive altered GC B cell differentiation during chronic infection even against unrelated antigens. However, the data also indicate that inflammatory circuits are likely linked to perception of antigen stimulation. Nevertheless, this study reveals a B cell-intrinsic program of transcriptional skewing in chronic viral infection that results in shunting out of the cyclic GC B cell process and early GC exit with consequences for antibody quality and hypergammaglobulinemia. These findings have implications for vaccination in individuals with pre-existing chronic infections where antibody responses are often ineffective and suggest that modulation of inflammatory pathways may be therapeutically useful to overcome impaired humoral immunity and foster affinity maturation during chronic viral infections.

 

References:

 

https://www.biorxiv.org/content/10.1101/849844v1

 

https://www.ncbi.nlm.nih.gov/pubmed/25656706

 

https://www.ncbi.nlm.nih.gov/pubmed/27653600

 

https://www.ncbi.nlm.nih.gov/pubmed/26912368

 

https://www.ncbi.nlm.nih.gov/pubmed/26799208

 

https://www.ncbi.nlm.nih.gov/pubmed/23001146

 

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

Once herpes simplex infects a person, the virus goes into hiding inside nerve cells, hibernating there for life, periodically waking up from its sleep to reignite infection, causing cold sores or genital lesions to recur. Research from Harvard Medical School showed that the virus uses a host protein called CTCF, or cellular CCCTC-binding factor, to display this type of behavior. Researchers revealed with experiments on mice that CTCF helps herpes simplex regulate its own sleep-wake cycle, enabling the virus to establish latent infections in the body’s sensory neurons where it remains dormant until reactivated. Preventing that latency-regulating protein from binding to the virus’s DNA, weakened the virus’s ability to come out of hiding.

 

Herpes simplex virus’s ability to go in and out of hiding is a key survival strategy that ensures its propagation from one host to the next. Such symptom-free latency allows the virus to remain out of the reach of the immune system most of the time, while its periodic reactivation ensures that it can continue to spread from one person to the next. On one hand, so-called latency-associated transcript genes, or LAT genes, turn off the transcription of viral RNA, inducing the virus to go into hibernation, or latency. On the other hand, a protein made by a gene called ICP0 promotes the activity of genes that stimulate viral replication and causes active infection.

 

Based on these earlier findings, the new study revealed that this balancing act is enabled by the CTCF protein when it binds to the viral DNA. Present during latent or dormant infections, CTCF is lost during active, symptomatic infections. The researchers created an altered version of the virus that lacked two of the CTCF binding sites. The absence of the binding sites made no difference in early-stage or acute infections. Similar results were found in infected cultured human nerve cells (trigeminal ganglia) and infected mice model. The researchers concluded that the mutant virus was found to have significantly weakened reactivation capacity.

 

Taken together, the experiments showed that deleting the CTCF binding sites weakened the virus’s ability to wake up from its dormant state thereby establishing the evidence that the CTCF protein is a key regulator of sleep-wake cycle in herpes simplex infections.

 

References:

 

https://www.ncbi.nlm.nih.gov/pubmed/29437926

 

https://hms.harvard.edu/news/viral-hideout?utm_source=Silverpop

 

https://www.ncbi.nlm.nih.gov/pubmed/30110885

 

https://www.ncbi.nlm.nih.gov/pubmed/30014861

 

https://www.ncbi.nlm.nih.gov/pubmed/18264117

 

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NEW Book #InfectiousDiseases #Immunology #StressSignaling #Therapeutics check https://www.amazon.com/dp/B075CXHY1B

Editor-in-Chief: Aviva Lev-Ari, PhD, RN

 

 

Includes FDA Approved Drugs for Infections and Infectious Diseases: Bacterial Infection, Viral Infection, Fungal Infection, Allergy-related Infections and Other, 1995 – 2016

VOLUME 2: covers the frontier of research on Infectious Diseases and the Human Immune System. The Immune Response, Disease Specific Immune Response, Immunodiagnostics and Immunotherapy, Immunotherapy and Autoimmunity,
Bacterial Infections, Bacteria Types, Antibactirial Therapeutics, FDA Approved Drugs for Infections and Infectious Diseases: Bacterial Infection, 1995 – 2016. Viral Infection: Virus Types, Antiviral Therapeutics, and FDA Approved Drugs for Infections and Infectious Diseases: Viral Infection, Fungal Infections, Allergy-related Infections, Other Infections,1995 – 2016,

VOLUME 3: covers the state of Science on the Historical Perspective of Immunology, Development of the Immune System, Signaling and Immunology, Cellular Immunity, Immunology and Inflammatory Response. Antibody-based Immunity, Vaccines and Microbiome, Immuno-Pharmaceutics, Cancer Immunotherapy, Immunomodulation and Neuro-Immunology.

Volume 2: Summary
The material that has been covered is a considerable material on the basic types of infections – bacterial, viral, and fungal, and diseases related to immune mechanisms. There has been a substantial coverage of the drugs and the manufacturers. This material brings to the discussion an international problem of drug resistance that applies much to bacteria, and a considerable amount of material on advances in drug development that takes into consideration protein structure and protein-protein interactions. The coverage of virus diseases brings to the forefront vaccines. However, in such cases as the influenza virus, a rapid genetic change of the virus makes the use of vaccines an issue for continuing revision.

Volume 3: Summary
The second volume is only concerned with the pathobiology of the inflammatory response, including sepsis, and it does not leave out hematopoiesis, and it lays out the difference between the B-clles and the T-cells that are related to the Toll receptor. Here we have looked closely at two immune disorders, Inflammatory Bowel Disease (Crohn’s Disease) and Rheumatoid Arthritis. Here we have discussed immunomodulation and signaling of the pathways involved, and the programmed cell death response. We have also covered the relationship of the immune response to autoimmune disorders and to cancer. The treatment of cancer now heavily leans toward the blocking of destructive processes in the immunomodulatory pathways.

Epilogue – Volume 2
Volume 2 has covered the most common bacterial and viral diseases that we find widely, or sporadically. It detailed the development of sepsis, and the immune response factor. The immune response involves local cellular invasion of lymphocytes related to initiation of T-cells and macrophages, and also the proteomic generated B-cell antibodies. These reactions are both local and systemic, as bacterial invasion is local and usually related to the tissue of residence (large intestine, oral, lung, genital). In the case of virus, the site of entry is often respiratory or by food intake, but these agents may rapidly become systemic. The other matter of the immune response is autoimmune, a reaction against the self. It is not entirely clear how this is initiated, but it has been related to failure to develop immunity in the prenatal or postnatal period. The only other possibility that might be considered would be by the mechanism of cell remodeling by an apoptotic related mechanism. The other chapters deal with therapeutics.

Epilogue – Volume 3
These two volumes have traversed a large knowledge-base. The first was directed largely at the well known bacterial, virus, fungal diseases, as well as autoimmunity. It specified recent FDA approved recommendations of pharmaceutics for these conditions. It also gives some attention to the immune response in inflammatory and autoimmune diseases, but not cancer. The second volume gives a concise history of development of Leukemias, Lymphomas pathology.

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Announcing our 10th e-Book on Amazon.com – 1st day, 9/4/2017

Editor-in-Chief: Aviva Lev-Ari, PhD, RN

 

On our Book Shelf on Amazon.com

WE ARE ON AMAZON.COM

https://www.amazon.com/s/ref=dp_byline_sr_ebooks_9?ie=UTF8&text=Aviva+Lev-Ari&search-alias=digital-text&field-author=Aviva+Lev-Ari&sort=relevancerank

http://www.amazon.com/dp/B00DINFFYC

http://www.amazon.com/dp/B018Q5MCN8

http://www.amazon.com/dp/B018PNHJ84

http://www.amazon.com/dp/B018DHBUO6

http://www.amazon.com/dp/B013RVYR2K

http://www.amazon.com/dp/B012BB0ZF0

http://www.amazon.com/dp/B019UM909A

http://www.amazon.com/dp/B019VH97LU

http://www.amazon.com/dp/B071VQ6YYK

https://www.amazon.com/dp/B075CXHY1B

 

The Immune System, Stress Signaling, Infectious Diseases and Therapeutic Implications: VOLUME 2: Infectious Diseases and Therapeutics and VOLUME 3: The Immune System and Therapeutics (Series D: BioMedicine & Immunology) Kindle Edition – on Amazon.com since 9/4/2017

by Larry H. Bernstein (Author), Aviva Lev-Ari (Author), Stephen J. Williams (Author), Demet Sag (Author), Irina Robu (Author), Tilda Barliya (Author), David Orchard-Webb (Author), Alan F. Kaul (Author), Danut Dragoi (Author), Sudipta Saha (Editor)

https://www.amazon.com/dp/B075CXHY1B

 

Product details

  • File Size:21832 KB
  • Print Length:3747 pages
  • Publisher:Leaders in Pharmaceutical Business Intelligence (LPBI) Group; 1 edition (September 4, 2017)
  • Publication Date:September 4, 2017
  • Sold by:Amazon Digital Services LLC
  • Language:English
  • ASIN:B075CXHY1B
  • Text-to-Speech: Enabled 
  • X-Ray: Not Enabled 
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  • Lending:Enabled
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Curator: Aviva Lev-Ari, PhD, RN

 

Transcriptomic Biomarkers to Discriminate Bacterial from Nonbacterial Infection in Adults Hospitalized with Respiratory Illness

Published online: 26 July 2017

URMC Researchers Developing New Tool to Fight Antibiotic Resistance

Goal is to Distinguish Between Viral and Bacterial Infections, Reduce Unnecessary Use of Antibiotics

Friday, July 28, 2017

“It’s extremely difficult to interpret what’s causing a respiratory tract infection, especially in very ill patients who come to the hospital with a high fever, cough, shortness of breath and other concerning symptoms,” said Ann R. Falsey, M.D., lead study author, professor and interim chief of the Infectious Diseases Division at UR Medicine’s Strong Memorial Hospital.

“My goal is to develop a tool that physicians can use to rule out a bacterial infection with enough certainty that they are comfortable, and their patients are comfortable, foregoing an antibiotic.”

Lead researcher Ann Falsey, M.D.

Ann R. Falsey, M.D.

Falsey’s project caught the attention of the federal government; she’s one of 10 semifinalists in the Antimicrobial Resistance Diagnostic Challenge, a competition sponsored by NIH and the Biomedical Advanced Research and Development Authority to help combat the development and spread of drug resistant bacteria. Selected from among 74 submissions, Falsey received $50,000 to continue her research and develop a prototype diagnostic test, such as a blood test, using the genetic markers her team identified.

SOURCE

https://www.urmc.rochester.edu/news/story/5108/urmc-researchers-developing-new-tool-to-fight-antibiotic-resistance.aspx

Lower respiratory tract infection (LRTI)

We enrolled 94 subjects who were microbiologically classified; 53 as “non-bacterial” and 41 as “bacterial”. RNAseq and qPCR confirmed significant differences in mean expression for 10 genes previously identified as discriminatory for bacterial LRTI. A novel dimension reduction strategy selected three pathways (lymphocyte, α-linoleic acid metabolism, IGF regulation) including eleven genes as optimal markers for discriminating bacterial infection (naïve AUC = 0.94; nested CV-AUC = 0.86). Using these genes, we constructed a classifier for bacterial LRTI with 90% (79% CV) sensitivity and 83% (76% CV) specificity. This novel, pathway-based gene set displays promise as a method to distinguish bacterial from nonbacterial LRTI.

https://www.nature.com/articles/s41598-017-06738-3#Sec8

IMAGE SOURCE

https://www.nature.com/articles/s41598-017-06738-3#Sec8

 

SOURCES

http://sciencemission.com/site/index.php?page=news&type=view&id=microbiology-virology%2Fnew-tool-to-distinguish&filter=8%2C9%2C10%2C11%2C12%2C13%2C14%2C16%2C17%2C18%2C19%2C20%2C27&redirected=1&redirected=1

https://www.urmc.rochester.edu/news/story/5108/urmc-researchers-developing-new-tool-to-fight-antibiotic-resistance.aspx

https://www.nature.com/articles/s41598-017-06738-3

Bacterial or Viral Infection? A New Study May Help Physicians …

 

Other related articles published in this Open Access Online Scientific Journal include the following:

Series D, VOLUME 2:

Infectious Diseases and Therapeutics

Author, Curator and Editor: Larry H Bernstein, MD, FCAP and CuratorSudipta Saha, PhD

 

Series D, VOLUME 3:

The Immune System and Therapeutics

Author, Curator and Editor: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/biomed-e-books/series-d-e-books-on-biomedicine/human-immune-system-in-health-and-in-disease/

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Signaling through the T Cell Receptor (TCR) Complex and the Co-stimulatory Receptor CD28

Curator: Larry H. Bernstein, MD, FCAP

 

 

New connections: T cell actin dynamics

Fluorescence microscopy is one of the most important tools in cell biology research because it provides spatial and temporal information to investigate regulatory systems inside cells. This technique can generate data in the form of signal intensities at thousands of positions resolved inside individual live cells. However, given extensive cell-to-cell variation, these data cannot be readily assembled into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. We have developed a method to enable comparison of imaging data from many cells and applied it to investigate actin dynamics in T cell activation. Antigen recognition in T cells by the T cell receptor (TCR) is amplified by engagement of the costimulatory receptor CD28. We imaged actin and eight core actin regulators to generate over a thousand movies of T cells under conditions in which CD28 was either engaged or blocked in the context of a strong TCR signal. Our computational analysis showed that the primary effect of costimulation blockade was to decrease recruitment of the activator of actin nucleation WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) and the actin-severing protein cofilin to F-actin. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics caused by costimulation blockade. Thus, we have developed and validated an approach to quantify protein distributions in time and space for the analysis of complex regulatory systems.

RELATED CONTENT

 

Triple-Color FRET Analysis Reveals Conformational Changes in the WIP-WASp Actin-Regulating Complex

 

RELATED CONTENT

T cell activation by antigens involves the formation of a complex, highly dynamic, yet organized signaling complex at the site of the T cell receptors (TCRs). Srikanth et al. found that the lymphocyte-specific large guanosine triphosphatase of the Rab family CRACR2A-a associated with vesicles near the Golgi in unstimulated mouse and human CD4+ T cells. Upon TCR activation, these vesicles moved to the immunological synapse (the contact region between a T cell and an antigen-presenting cell). The guanine nucleotide exchange factor Vav1 at the TCR complex recruited CRACR2A-a to the complex. Without CRACR2A-a, T cell activation was compromised because of defective calcium and kinase signaling.

More than 60 members of the Rab family of guanosine triphosphatases (GTPases) exist in the human genome. Rab GTPases are small proteins that are primarily involved in the formation, trafficking, and fusion of vesicles. We showed that CRACR2A (Ca2+ release–activated Ca2+ channel regulator 2A) encodes a lymphocyte-specific large Rab GTPase that contains multiple functional domains, including EF-hand motifs, a proline-rich domain (PRD), and a Rab GTPase domain with an unconventional prenylation site. Through experiments involving gene silencing in cells and knockout mice, we demonstrated a role for CRACR2A in the activation of the Ca2+ and c-Jun N-terminal kinase signaling pathways in response to T cell receptor (TCR) stimulation. Vesicles containing this Rab GTPase translocated from near the Golgi to the immunological synapse formed between a T cell and a cognate antigen-presenting cell to activate these signaling pathways. The interaction between the PRD of CRACR2A and the guanidine nucleotide exchange factor Vav1 was required for the accumulation of these vesicles at the immunological synapse. Furthermore, we demonstrated that GTP binding and prenylation of CRACR2A were associated with its localization near the Golgi and its stability. Our findings reveal a previously uncharacterized function of a large Rab GTPase and vesicles near the Golgi in TCR signaling. Other GTPases with similar domain architectures may have similar functions in T cells.

 

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Phosphorylation-dependent interaction between antigenic peptides and MHC class I

Curator: Larry H. Bernstein, MD, FCAP

 

 

Phosphorylation-dependent interaction between antigenic peptides and MHC class I: a molecular basis for the presentation of transformed self.

Nat Immunol. 2008 Nov;9(11):1236-43.    http://dx.doi.org:/10.1038/ni.1660.  Epub 2008 Oct 5.
Protein phosphorylation generates a source of phosphopeptides that are presented by major histocompatibility complex class I molecules and recognized by T cells. As deregulated phosphorylation is a hallmark of malignant transformation, the differential display of phosphopeptides on cancer cells provides an immunological signature of ‘transformed self’. Here we demonstrate that phosphorylation can considerably increase peptide binding affinity for HLA-A2. To understand this, we solved crystal structures of four phosphopeptide-HLA-A2 complexes. These identified a novel peptide-binding motif centered on a solvent-exposed phosphate anchor. Our findings indicate that deregulated phosphorylation can create neoantigens by promoting binding to major histocompatibility complex molecules or by affecting the antigenic identity of presented epitopes. These results highlight the potential of phosphopeptides as novel targets for cancer immunotherapy.
Figure 1
Bioinformatic characterization of the HLA-A2–restricted phosphopeptide repertoire. (a) Distribution of phosphorylated residues among naturally processed (A2 phosphopeptide) and predicted HLA-A2 binding phosphopeptides (Phosphosite, EMBL). The frequency of phosphorylated residues at each position is displayed for naturally processed HLA-A2 associated phosphopeptides, and for peptides in EMBL and Phosphosite datasets that contain phosphorylation sites and are predicted, according to criteria described in Methods, to bind HLA-A2. (b) Representation of positively charged residues (Arg or Lys) at P1 among naturally processed HLA-A2 associated phosphopeptides, phosphopeptides from the EMBL or Phosphosite datasets that are predicted to bind HLA-A2 and contain a p-Ser residue at the P4 position, and datasets of naturally processed non-phosphorylated peptides (B-LCL) and known HLA-A2 binding peptides (Immune Epitope). Selection criteria for the latter two datasets are described in Methods. * = P<0.001, NS= not significant. (c, d) Representation of subdominant residues at the P2 anchor position (c) and the PC (P9) position (d) in naturally processed HLA-A2 associated phosphopeptides and in datasets of naturally processed non-phosphorylated peptides and known HLA-A2 binding peptides.
Changes in protein expression or metabolism due to intracellular infection or cellular transformation modify the repertoire of peptides generated and therefore displayed by class I MHC molecules, resulting in presentation of “altered self” to the immune system. T cell receptor (TCR)-mediated recognition of specific MHC-bound peptides by CD8 T lymphocytes results in cytolytic activity and release of pro-inflammatory cytokines, which are key components of anti-viral and anti-tumor immunity. Evidence suggests that peptides containing post-translational modifications (PTM), including deamidation, cysteinylation, glycosylation, and phosphorylation, contribute to the pool of MHC-bound peptides presented at the cell surface and represent potential targets for T cell recognition2. Indeed, the majority of naturally occurring PTM-bearing peptides defined to date can be discriminated from their unmodified homologs specifically by T cells2-4.  …..
Recent studies have highlighted protein phosphorylation as a process with the capacity to generate unique peptides bound to class I MHC molecules. Significant numbers of different phosphorylated peptides are presented by several HLA-A and HLA-B alleles that are prevalent in humans3,4, demonstrating their widespread potential as antigens. Moreover, CD8+ T lymphocytes recognize these phosphopeptides in a manner that is both peptide sequence-specific and phosphate-dependent3, 4. Thus, phosphopeptides can be immunologically distinguished from their non-phosphorylated counterparts. Consistent with their presentation by class I MHC molecules, most phosphorylated peptides are derived from proteins that function intracellularly, and processing of both model and naturally occurring phosphopeptides is dependent on transport into the endoplasmic reticulum (ER) by transporter associated with antigen processing (TAP)3, 5. Furthermore, rapid degradation by the proteasome, a process that regulates the activity of many transcription factors, cell growth modulators, signal transducers and cell cycle proteins6-8, is frequently dependent on target protein phosphorylation9-11. ….
Phosphopeptide antigens are of significant therapeutic interest because deregulation of protein kinase activity, normally tightly controlled, is one of the hallmarks of malignant transformation and is thought to contribute directly to oncogenic signaling pathways involved in cell growth, differentiation and survival13-15. In addition, mutation-induced deregulation of a limited number of critical kinases can often lead to activation of several signaling cascades and increases in the extent of protein phosphorylation within the cell16-18. These considerations strongly suggest that alterations in protein phosphorylation during malignancy represent a distinctive immunological signature of “transformed self”. Consistent with this notion, the phosphopeptides presented by HLA-A*0201….

Nα-Terminal Acetylation for T Cell Recognition: Molecular Basis of MHC Class I–Restricted Nα-Acetylpeptide Presentation

As one of the most common posttranslational modifications (PTMs) of eukaryotic proteins, Nα-terminal acetylation (Nt-acetylation) generates a class of Nα-acetylpeptides that are known to be presented by MHC class I at the cell surface. Although such PTM plays a pivotal role in adjusting proteolysis, the molecular basis for the presentation and T cell recognition of Nα-acetylpeptides remains largely unknown. In this study, we determined a high-resolution crystallographic structure of HLA (HLA)-B*3901 complexed with an Nα-acetylpeptide derived from natural cellular processing, also in comparison with the unmodified-peptide complex. Unlike the α-amino–free P1 residues of unmodified peptide, of which the α-amino group inserts into pocket A of the Ag-binding groove, the Nα-linked acetyl of the acetylated P1-Ser protrudes out of the groove for T cell recognition. Moreover, the Nt-acetylation not only alters the conformation of the peptide but also switches the residues in the α1-helix of HLA-B*3901, which may impact the T cell engagement. The thermostability measurements of complexes between Nα-acetylpeptides and a series of MHC class I molecules derived from different species reveal reduced stability. Our findings provide the insight into the mode of Nα-acetylpeptide–specific presentation by classical MHC class I molecules and shed light on the potential of acetylepitope-based immune intervene and vaccine development.

Produced by Ag processing and proteasomal degradation of intracellular proteins, polypeptides serve as CTL epitopes presented by MHC class I molecules, which play a critical role in cellular immunity (1). Eukaryotic proteins bearing various posttranslational modifications (PTMs) can generate a group of modified Ags, which contribute to a special repertoire of MHC-associated peptides presented at the cell surface as potential targets for TCR-mediated recognition. A modified peptide may become a new Ag because of the distinguished antigenicity compared with its unmodified homolog. A variety of natural peptide Ags containing modification have been observed that can be immunologically discriminated by T cells from their unmodified homologs as “altered self” (2). Thus, the significance of PTMs on epitopes and the application of modified peptides in vaccine development for immunotherapy against cancer and autoimmune diseases have been increasingly appreciated (3, 4).

The molecular bases of the presentation of peptides with several PTMs by MHC class I molecules have been successfully explicated. For instance, the formyl group on an Nt-formylated peptide binds to the bottom of the peptide-binding groove of H2-M3 (5); both the glycan and the phosphate moieties of the central region of the glycopeptides (6, 7) and the phosphopeptides (8, 9), respectively, are exposed to enable TCR binding, and the deimination (citrullination) of arginine on a peptide presented by two HLA-B27 subtypes induces distinct peptide conformations (10).

Nα-terminal acetylation (Nt-acetylation) is one of the most common PTMs, occurring on the vast majority of eukaryotic proteins. In humans, >80% of the different varieties of intracellular proteins are irreversibly Nt-acetylated by Nα-acetyltransferases, often after the removal of the initiator methionine. Only a subset of the penultimate residues (Ala, Ser, Thr, Cys, and Val) or the retained initiator methionine can be acetylated at the α-amino (NH2) groups (11). A recent study found that acetylated N-terminal residues of eukaryotic proteins act as specific degradation signals (Ac-N-degrons) that are recognized by specific ubiquitin ligases (12). A subsequent systematic analysis demonstrated that Nt-acetylation can also represent an early determining factor in the cellular sorting for prevention of protein targeting to the secretory pathway (13). These findings suggested that Nt-acetylation–mediated inhibition of secretion could contribute to the retention of proteins in the cytosol where they may subsequently be ubiquitinylated through the specific recognition of their Ac-N-degrons and thereby generating Nt-acetylated proteasomal digestion products (14). Hence, these Nt-acetylated polypeptides in the form of MHC-associated neoantigens stand a good chance to be recognized by T cells. This has indeed been illuminated in an Nt-acetylated MHC class II–restricted peptide derived from myelin basic protein, which stimulates murine T cells to elicit experimental autoimmune encephalomyelitis, whereas the nonacetylated form does not (15). A structural study subsequently suggested that the Nt-acetylation of this peptide is essential for MHC class II binding (16).

For MHC class I, the first Nt-acetylated natural ligand was identified more than a decade ago (17). However, the mode of interaction of this acetylated peptide with class I molecules remained largely enigmatic. To understand this, we determined the crystal structures of a naturally occurring Nt-acetylated self-peptide (NAc-SL9) and two nonmodified variants (SL9 and HL8), respectively, in complex with HLA-B*3901. Taken together with the thermostability analyses of Nα-acetylpeptides complexed with a series of class I molecules of human and murine origin, we elucidated that Nt-acetylation exerts a destabilizing effect on peptide–MHC (pMHC) complex, thereby influencing TCR recognition.

……

Our results here provide the structural and thermodynamic insights into the presentation of Nt-acetylated peptides by MHC class I molecules. The structure of the Nα-acetylpeptide in complex with HLA-B*3901 outlines a molecular interpretation of the reduced stability of MHC class I–bound Nt-acetylated peptides and also highlights a potential influence of Nt-acetylation on antigenic identity and T cell recognition. In addition, the structure elucidation of HLA-B*3901, the predominant B39 subtype, also is valuable in studying immune diseases associated with this MHC allele.

In a previous report, the Nt-formyl group on an Nt-formylated peptide binds to the bottom of the peptide-binding groove of the murine MHC class I H2-M3 playing an anchoring role for MHC class I binding (Supplemental Fig. 2A) (5). In our study, the methyl and carbonyl groups of the acetyl are rotated upwards like two arms that push the peptide-binding groove open (Fig. 2G, Supplemental Fig. 2B), thereby altering its immunogenicity at the expense of the pMHC stability. The thermostability we tested from seven human and one murine complexes indicates a general feature of Nα-acetylpeptide in weakening the binding affinity to MHC class I, which could be revealed by the gel-filtration chromatography of pMHC refolding assays as well (Supplemental Fig. 3). Their instability would partially explain why, as yet, such epitopes are rarely found. Within N-terminal residues of eukaryotic proteins, Ser is the most frequently acetylated in vivo (11). The Ala, Thr, Cys, and Val residues can also be Nt-acetylated and have small side chains like Ser. Thus, the rotation of P1 residues observed in the pHLA-B*3901 complex with an acetylated P1-Ser could very well be a general mode in Nα-acetylpeptide binding. In contrast, the long side chain of Met precludes it from being rotated into pocket A, but a certain reorientation is presumed to take place in the acetylated P1-Met based on the thermal instability (Fig. 6H). Besides the accommodation of the acetyl moiety, Nt-acetylation is presumed to decrease the stability of the pHLA-B*3901 complex as a result of the conformational switch of the Arg62. Arg62 in the α1-helix is largely conserved in almost all HLA-B and -C allotypes (Table V). For other HLA class I (Table V, Fig. 8), the long charged side chains of the residues in position 62 (Glu62 of A24 and Gln62 of A11 and so on) also may interact with the acetyl. Hence, the residue in position 62 plays a key role in the interaction between acetyl group and the H chain, which may influence not only the Nα-acetylpeptide binding to HLA molecules but also the TCR docking.

The discoveries that intracellular proteins with Ac-N-degrons are inhibited from being secreted (13) and then are degraded via ubiquitylation (12) raise many questions on the biological significance of acetylation-mediated proteolysis (14). The Nt-acetylated peptides with the size of MHC class I ligands (8–11 aa) as neoepitopes for CD8+ T cells, represent one of the possible roles of the Nt-acetylated digestion products. The vast armory of intracellular proteins that are frequently Nt-acetylated can create a large pool of Nα-acetylpeptides for Ag presentation and T cell surveying. The Nt-acetylation potentially impacts the TCR-MHC interaction in three different aspects: 1) the direct interaction of the solvent-exposed acetyl moiety; 2) the altered conformation of the central region of the peptide main chain; and 3) the conformational switches of the MHC residues. The Nt-acetylation creation of a distinctive pMHC landscape and participation in a potential binding element for TCR engagement described in our results highlights needs for further investigation into the Nα-acetylpeptide–specific TCR repertoires.  ……

see…J Immunol 2014; 192:5509-5519   http://dx.doi.org:/10.4049/jimmunol.1400199   http://www.jimmunol.org/content/192/12/5509

Supplementary http://www.jimmunol.org/content/suppl/2014/05/14/jimmunol.1400199.DCSupplemental.html
References http://www.jimmunol.org/content/192/12/5509.full#ref-list-1

 

The Cellular Redox Environment Alters Antigen Presentation*

Jonathan A. Trujillo,§12Nathan P. Croft,1Nadine L. Dudek,1Rudragouda ChannappanavarAlex TheodossisAndrew I. Webb,…., Jamie Rossjohn,‡‡,§§5Stanley Perlman,§6 and Anthony W. Purcell,7
The Journal of Biological Chemistry 289; 27979-27991.
http://dx.doi.org:/10.1074/jbc.M114.573402

Capsule

Background: Modification of cysteine residues, including glutathionylation, commonly occurs in peptides bound to and presented by MHC molecules.

Results: Glutathionylation of a coronavirus-specific T cell epitope results in diminished CD8 T cell recognition.

Conclusion: Cysteine modification of a T cell epitope negatively impacts the host immune response.

Significance: Cross-talk between virus-induced oxidative stress and the T cell response probably occurs, diminishing host cell recognition of infected cells.

Cysteine-containing peptides represent an important class of T cell epitopes, yet their prevalence remains underestimated. We have established and interrogated a database of around 70,000 naturally processed MHC-bound peptides and demonstrate that cysteine-containing peptides are presented on the surface of cells in an MHC allomorph-dependent manner and comprise on average 5–10% of the immunopeptidome. A significant proportion of these peptides are oxidatively modified, most commonly through covalent linkage with the antioxidant glutathione. Unlike some of the previously reported cysteine-based modifications, this represents a true physiological alteration of cysteine residues. Furthermore, our results suggest that alterations in the cellular redox state induced by viral infection are communicated to the immune system through the presentation of S-glutathionylated viral peptides, resulting in altered T cell recognition. Our data provide a structural basis for how the glutathione modification alters recognition by virus-specific T cells. Collectively, these results suggest that oxidative stress represents a mechanism for modulating the virus-specific T cell response.

Antigen Presentation     Antigen Processing     Glutathionylation     Mass Spectrometry (MS)     Oxidation-Reduction (Redox)     Redox Regulation     T-cell     Viral Immunology

Small fragments of proteins (peptides) derived from both intracellular and extracellular sources are displayed on the surface of cells by molecules encoded within the major histocompatibility complex (MHC). These peptides are recognized by T lymphocytes and provide the immune system with a surveillance mechanism for the detection of pathogens and cancer cells. The fidelity with which antigen presentation communicates changes in the intracellular proteome is critical for immune surveillance. Not only do antigens expressed at vastly different abundances need to be represented within the array of peptides selected and presented at the cell surface (collectively termed the immunopeptidome (1, 2)), but changes in their post-translational state also need to be conveyed within this complex mixture of peptides. For example, changes in antigen phosphorylation have been linked to cancer, and the presentation of phosphorylated peptides has been shown to communicate the cancerous state of cells to the immune system (36). Other types of post-translational modification play a central role in the pathogenesis of autoimmune diseases (7), such as arginine citrullination in arthritis (810), deamidation of glutamine residues in wheat proteins in celiac disease (1115), and cysteine oxidation in type 1 diabetes (16, 17). Cysteine is predicted to be present in up to 14% of potential T cell epitopes based on its prevalence in various pathogen and host proteomes (18). However, reports of cysteine-containing epitopes are much less frequent due to technical difficulties associated with synthesis and handling of cysteine-containing peptides and their subsequent avoidance in many epitope mapping studies (19). Cysteine can be modified in numerous ways, including cysteinylation (the disulfide linkage of free cysteine to peptide or protein cysteine residues), oxidation to cysteine sulfenic (oxidation), sulfinic (dioxidation) and sulfonic acids (trioxidation), S-nitrosylation, and S-glutathionylation. Such modifications may occur prior to or during antigen processing; however, the role of cysteine modification in T-cell-mediated immunity has not been systematically addressed.

In addition to constitutive presentation of a subset of oxidatively modified peptides, it is anticipated that changes in the proportion of these ligands will occur upon infection because oxidative stress, triggering of the unfolded protein response, and modulation of host cell synthesis by the virus are hallmarks of this process (2027). For example, host cell stress responses modulate expression, localization, and function of Toll-like receptors, a key event in the initiation of the immune response (28). Oxidative stress would also be predicted to affect protein function through post-translational modification of amino acids, such as cysteine. Indeed, because of the reactive nature of cysteine and the requirements for cells to regulate the redox state of proteins to maintain function, a number of scavenging systems for redox-reactive intermediates exist. The tripeptide glutathione (GSH) is one of the key intracellular antioxidants, acting as a scavenger for reactive oxygen species. Reduced GSH is equilibrated with its oxidized form, GSSG, with normal cytosolic conditions being that of the reduced state in a ratio of ∼50:1 (GSH/GSSG) (29). Modification of proteins and peptides with GSH (termed S-glutathionylation) occurs following reaction of GSSG with the thiol group of cysteine in a reaction catalyzed by the detoxifying enzyme, glutathione S-transferase (GST). A variety of cellular processes and signaling pathways, such as the induction of innate immunity, apoptosis, redox homeostasis, and cytokine production, are modulated by this GST-catalyzed post-translational modification (3032). S-Glutathionylation can eventuate via oxidative stress, whereby the intracellular levels of GSSG increase.

Given that viruses are known to induce oxidative stress (3335), the intracellular environment of viral infection may lead to an increase inS-glutathionylated cellular proteins and viral antigens. For instance, HSV infection induces an early burst of reactive oxygen species, resulting in S-glutathionylation of TRAF family members, which in turn is linked to downstream signaling and interferon production (36). The potential for modification of viral antigens subsequent to reactive oxygen species production is highlighted by S-glutathionylation of several retroviral proteases, leading to host modulation of protease function (37). Indeed large scale changes in protein S-glutathionylation are observed in HIV-infected T cell blasts (38), suggesting that functional modulation of both host and viral proteins occurs via this mechanism. Whether these S-glutathionylated proteins inhibit or enhance immune responses to the unmodified epitope or generate novel T-cell epitopes that are subsequently recognized by the adaptive immune system is unclear.

Here, we investigate the frequency of modification of cysteine-containing MHC-bound peptides by interrogating a large database of naturally processed self-peptides derived from B-lymphoblastoid cells, murine tissues, and cytokine-treated cells. In addition, the functional consequences of Cys modification of T cell epitopes was investigated using an established model of infection that involves an immunodominant cysteine-containing epitope derived from a neurotropic strain of mouse hepatitis virus, strain JHM (JHMV)8(3941). We describe S-glutathionylation of this viral T cell epitope and the functional and structural implications of redox-modulated antigen presentation. Collectively our studies suggest that S-glutathionylation plays a key, previously unappreciated role in adaptive immune recognition.

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CD-4 Therapy for Solid Tumors

Curator: Larry H. Bernstein, MD, FCAP

 

CD4 T-cell Immunotherapy Shows Activity in Solid Tumors

Alexander M. Castellino, PhD

http://www.medscape.com/viewarticle/862095

For the first time, treatment with genetically engineered T-cells has used CD4 T-cells instead of the CD8 T-cells, which are used in the chimeric antigen receptor (CAR) T-cell approach. Early data suggest that this CD4 T-cell approach has activity against solid tumors, whereas the CAR T-cell approach so far has achieved dramatic success in hematologic malignancies.

In the new approach, CD4 T-cells were genetically engineered to target MAGE-A3, a protein found on many tumor cells. The treatment was found to be safe in patients with metastatic cancers, according to data from a phase 1 clinical study presented here at the American Association for Cancer Research (AACR) 2016 Annual Meeting.

“This is the first trial testing an immunotherapy using genetically engineered CD4 T-cells,” senior author Steven A. Rosenberg, MD, PhD, chief of the Surgery Branch at the National Cancer Institute (NCI), told Medscape Medical News.

Most approaches use CD8 T-cells. Although CD8 T-cells are known be cytotoxic and CD4 T-cells are normally considered helper cells, CD4 T-cells can induce tumor regression, he said.

Louis M. Weiner, MD, director of the Lombardi Comprehensive Cancer Center at Georgetown University, in Washington, DC, indicated that in contrast with CAR T-cells, these CD4 T-cells target proteins on solid tumors. “CAR T-cells are not tumor specific and do not target solid tumors,” he said.

Engineering CD4 Cells

Immunotherapy with engineered CD4 T-cells was personalized for each patient whose tumors had not responded to or had recurred following treatment with least one standard therapy. The immunotherapy was specific for patients in whom a specific human leukocyte antigen (HLA) — HLA-DPB1*0401 — was found to be expressed on their cells and whose tumors expressed MAGE-A3.

MAGE-A3 belongs to a class of proteins expressed during fetal development. The expression is lost in normal adult tissue but is reexpressed on tumor cells, explained presenter Yong-Chen William Lu, PhD, a research fellow in the Surgery Branch of the NCI.

Targeting MAGE-A3 is relevant, because it is frequently expressed in a variety of cancers, such as melanoma and urothelial, esophageal, and cervical cancers, he pointed out.

 Researchers purified CD4 T-cells from the peripheral blood of patients. Next, the CD4 T-cells were genetically engineered with a retrovirus carrying the T-cell receptor (TCR) gene that recognizes MAGE-A3. The modified cells were grown ex vivo and were transferred back into the patient.

Clinical Results

Dr Lu presented data for 14 patients enrolled into the study: eight patients received cell doses from 10 million to 30 billion cells, and six patients received up to 100 billion cells.

This was similar to a phase 1 dose-finding study, except the researchers were seeking to determine the maximum number of genetically engineered CD4 T-cells that a patient could safely receive.

One patient with metastatic cervical cancer, another with metastatic esophageal cancer, and a third with metastatic urothelial cancer experienced partial objective responses. At 15 months, the response is ongoing in the patient with cervical cancer; after 7 months of treatment, the response was durable in the patient with urothelial cancer; and a response lasting 4 months was reported for the patient with esophageal cancer.

Dr Lu said that a phase 2 trial has been initiated to study the clinical responses of this T-cell receptor therapy in different types of metastatic cancers.

In his discussion of the paper, Michel Sadelain, MD, of the Memorial Sloan Kettering Cancer Center, New York City, said, “Although therapy with CD4 cells has been evaluated using endogenous receptor, this is the first study using genetically engineered CD4 T-cells.”

Although the study showed that therapy with genetically engineered T-cells is safe and efficacious at least in three patients, the mechanism of cytotoxicity remains unclear, Dr Sadelain indicated.

Comparison With CAR T-cells

CAR T-cells act in much the same way. CARs are chimeric antigen receptors that have an antigen-recognition domain of an antibody (the V region) and a “business end,” which activates T-cells. In this case, CD8 T-cells from the patients are used to genetically engineer T-cells ex vivo. In the majority of cases, dramatic responses have been seen in hematologic malignancies.

CARs, directed against self-proteins, result in on-target, off-tumor effects, Gregory L. Beatty, MD, PhD, assistant professor of medicine at the University of Pennsylvania, in Philadelphia, indicated when he reported the first success story of CAR T-cells in a solid pancreatic cancer tumor.

Side effects of therapy with CD4 T-cells targeting MAGE-A3 were different and similar to side effects of chemotherapy, because patients received a lymphodepleting regimen of cyclophosphamide and fludabarine. Toxicities included high fever, which was experienced by the majority of patients (12/14). The fever lasted 1 to 2 weeks and was easily manageable.

High levels of the cytokine interleukin-6 (IL-6) were detected in the serum of all patients after treatment. However, the elevation in IL-6 levels was not considered to be a cytokine release syndrome, because no side effects occurred that correlated with the syndrome, Dr Liu indicated.

He also indicated that future studies are planned that will employ genetically engineered CD4 T-cells in combination with programmed cell death protein 1–blocking antibodies.

This study was funded by Intramural Research Program of the National Institutes of Health. The NCI’s research and development of T-cell receptor therapy targeting MAGE-A3 are supported in part under a cooperative research and development agreement between the NCI and Kite Pharma, Inc. Kite has an exclusive, worldwide license with the NIH for intellectual property relating to retrovirally transduced HLA-DPB1*0401 and HLA A1 T-cell receptor therapy targeting MAGE-A3 antigen. Dr Lu and Dr Rosenberg have disclosed no relevant financial relationships.

American Association for Cancer Research (AACR) 2016 Annual Meeting: Abstract CT003, presented April 17, 2016.

 

Searches Related to immunotherapy using genetically engineered CD4 T-cells

 

Genetic engineering of T cells for adoptive immunotherapy

To be effective for the treatment of cancer and infectious diseases, T cell adoptive immunotherapy requires large numbers of cells with abundant proliferative reserves and intact effector functions. We are achieving these goals using a gene therapy strategy wherein the desired characteristics are introduced into a starting cell population, primarily by high efficiency lentiviral vector-mediated transduction. Modified cells are then expanded using ex vivo expansion protocols designed to minimally alter the desired cellular phenotype. In this article, we focus on strategies to (1) dissect the signals controlling T cell proliferation; (2) render CD4 T cells resistant to HIV-1 infection; and (3) redirect CD8 T cell antigen specificity.
Adoptive T cell therapy is a form of transfusion therapy involving the infusion of large numbers of T cells with the aim of eliminating, or at least controlling, malignancies or infectious diseases. Successful applications of this technique include the infusion of CMV-or EBVspecific CTLs to protect immunosuppressed patients from these transplantation-associated diseases [1,2]. Furthermore, donor lymphocyte infusions of ex vivo-expanded allogeneic T cells have been used to successfully treat hematological malignancies in patients with relapsed disease following allogeneic hematopoietic stem cell transplant [3]. However, in many other malignancies and chronic viral infections such as HIV-1, adoptive T cell therapy has achieved inconsistent and/or marginal successes. Nevertheless, there are compelling reasons for optimism on this strategy. For example, the existence of HIV-positive elite non-progressors [4], as well as the correlation between the presence of intratumoral T cells and a favorable prognosis in malignancies such as ovarian [5,6] and colon carcinoma [7,8], provides in vivo evidence for the critical role of the immune system in controlling both HIV and cancer.
The key to successful adoptive immunotherapy strategies appears to consist of (1) using the “right” T cell type(s) and (2) obtaining therapeutically effective numbers of these cells without compromising their effector functions or their ability to engraft within the host. This article is focused on strategies employed in our laboratory to generate the “right” cell through genetic engineering approaches, with an emphasis on redirecting the antigen specificity of CD8 T cells, and rendering CD4 T cells resistant to HIV-1 infection. The article by Paulos et al. describes the evolving process of how to best obtain therapeutically effective numbers of the “right” cells by optimizing ex vivo cell expansion strategies.
Our laboratory’s overall strategy and flow plan for development and evaluation of engineered T cells is depicted in Fig. 1. We work almost exclusively with primary human T cells; little or no work is performed with conventional established cell lines. Thus, we benefit substantially from our close association with the UPenn Human Immunology Core. The Core performs leukaphereses on healthy donors 2–3 times a week, and provides purified peripheral blood mononuclear cell subsets, ensuring a constant influx of fresh human T cells into our laboratory. We have extensive experience in developing both bead- and cell-based artificial antigen presenting cells (aAPCs), as described in detail in the article by Paulos et al. The ability to genetically modify T cells at high efficiency is critical for virtually every project within the laboratory. We have adapted the lentiviral vector system described by Dull [15] for most, but not all, of the engineering applications in our laboratory.
CD4 T cells are the primary target of HIV-1, and decreasing CD4 T cell numbers is a hallmark of advancing HIV-1 disease [34]. Thus, strategies that protect CD4 T cells from HIV-1 infection in vivo would conceivably provide sufficient immunological help to control HIV-1 infection. Our early observations that CD3/CD28 costimulation resulted in improved ex vivo expansion of CD4 T cells from both healthy and HIV-infected donors, as well as enhanced resistance to HIV-1 infection [35,36], ultimately led to the first-in-human trial of lentiviral vector-modified CD4 T cells [37]. In this trial, CD4 T cells from HIV-positive subjects who had failed antiretroviral therapy were transduced with a lentiviral vector encoding an antisense RNA that targeted a 937 bp region in the HIV-1 envelope gene. Preclinical studies demonstrated that this antisense region, directed against the HIV-1NL4-3 envelope, provided robust protection from a broad range of both R5-and X4-tropic HIV-1 isolates [38]. One year after administration of a single dose of the gene-modified cells, four of the five enrolled patients had increased peripheral blood CD4 T cell counts, and in one subject, a 1.7 log decrease in viral load was observed. Finally, in two of the five patients, persistence of the gene-modified cells was detected one year post-infusion.
Since its identification as the primary co-receptor involved in HIV transmission, CCR5 has attracted considerable attention as a target for HIV therapy [42,43]. Indeed, “experiments of nature” have shown that individuals with a homozygous CCR5 Δ32 deletion are highly resistant to HIV-1 infection. Thus, we hypothesized that knocking out the CCR5 locus would generate CD4 T cells permanently resistant to infection by R5 isolates of HIV-1. To test this hypothesis we took advantage of zinc-finger nuclease (ZFN) technology [44]. ZFNs introduce sequencespecific double-strand DNA breakage, which is imperfectly repaired by non-homologous endjoining. This results in the permanent disruption of the genomic target, a process termed genome editing (Fig. 3).
Genetic modification of T cells to redirect antigen specificity is an attractive strategy compared to the lengthy process of growing T cell lines or CTL clones for adoptive transfer. Genetically modified, adoptively transferred T cells are capable of long-term persistence in humans [37, 46,47], demonstrating the feasibility of this approach. When compared to the months it can take to generate an infusion dose of antigen-specific CTL lines or clones from a patient, a homogeneous population of redirected antigen-specific cells can be expanded to therapeutically relevant numbers in about two weeks [3]. Several strategies are being explored to bypass the need to expand antigen-specific T cells for adoptive T cell therapy. The approaches currently studied in our laboratory involve the genetic transfer of chimeric antigen receptors and supraphysiologic T cell receptors.
Chimeric antigen receptors (CARs or T-bodies) are artificial T cell receptors that combine the extracellular single-chain variable fragment (scFv) of an antibody with intracellular signaling domains, such as CD3ζ or Fc(ε)RIγ [48–50]. When expressed on T cells, the receptor bypasses the need for antigen presentation on MHC since the scFv binds directly to cell surface antigens. This is an important feature, since many tumors and virus-infected cells downregulate MHCI, rendering them invisible to the adaptive immune system. The high-affinity nature of the scFv domain makes these engineered T cells highly sensitive to low antigen densities. In addition, new chimeric antigen receptors are relatively easy to produce from hybridomas. The key to this approach is the identification of antigens with high surface expression on tumor cells, but reduced or absent expression on normal tissues.  Since one can redirect both CD4 and CD8 T cells, the T-body approach to immunotherapy represents a near universal “off the shelf” method to generate large numbers of antigen-specific helper and cytotoxic T cells.
Many T-bodies targeting diverse tumors have been developed [51], and four have been evaluated clinically [52–55]. Three of the four studies were characterized by poor transgene expression and limited T-body engraftment. However, in a study of metastatic renal cell carcinoma using a T-body directed against carbonic anhydrase IX [55], T-body-expressing cells were detectable in the peripheral blood for nearly 2 months post-administration.
The major goals in the T-body field currently are to optimize their engraftment and maximize their effector functions. Our laboratory is addressing both problems simultaneously through an in-depth study of the requirements for T-body activation. We hypothesize that their limited persistence is due to incomplete cell activation due to the lack of costimulation. While naïve T cells depend on costimulation through CD28 ligation to avoid anergy and undergo full activation in response to antigen, it is recognized that effector cells also require costimulation to properly proliferate and produce cytokines [56]. Previous studies have shown that providing CD28 costimulation is crucial for the antitumoral function of adoptively transferred T cells and T-bodies [57–59]. Unlike conventional T cell activation, which requires two discrete signals, T-bodies can be engineered to provide both costimulation and CD3 signaling through one binding event.
A different approach for redirecting specificity to T cells for adoptive immunotherapy involves the genetic transfer of full-length TCR genes. A T cell’s specificity for its cognate antigen is solely determined by its TCR. Genes encoding the α and β chains of a T cell receptor (TCR) can be isolated from a T cell specific for the antigen of interest and restricted to a defined HLA allele, inserted into a vector, and then introduced into large numbers of T cells of individual patients that share the restricting HLA allele as well as the targeted antigen. In 1999, Clay and colleagues from Rosenberg’s group at the National Cancer Institute were the first to report the transfer of TCR genes via a retroviral vector into human lymphocytes and to show that T cells gained stable reactivity to MART-1 [67]. To date, many others have shown that the same approach can be used to transfer specificity for multiple viral and tumor associated antigens in mice and human systems. These T cells gain effector functions against the transferred TCR’s cognate antigen, as defined by proliferation, cytokine production, lysis of targets presenting the antigen, trafficking to tumor sites in vivo, and clearance of tumors and viral infection.
In 2006, Rosenberg’s group redirected patients’ PBLs with the naturally occurring, MART-1- specific TCR reported in 1999 by Clay. In the first clinical trial to test TCR-transfer immunotherapy, these modified T cells were infused into melanoma patients [68]. While the transduced T cells persisted in vivo, only two of the 17 patients had an objective response to this therapy. One issue revealed by the study was the poor expression of the transgenic TCRs by the transferred T cells. Nonetheless, the results from this trial showed the potential of TCR transfer immunotherapy as a safe form of therapy for cancer and highlighted the need to optimize such therapy to attain maximum potency.
The adoptive immunotherapy field is advancing by a tried-and-true method: learning from disappointments and moving forward. Our ability to fully realize the therapeutic potential of adoptive T cell therapy is tied to a more complete understanding of how human T cells receive signals, kill targets, and modulate effective immune responses. Our goal is to perform labbased experiments that provide insight into how primary T cells function in a manner that will facilitate and enable adoptive T cell therapy clinical trials. Our ability to efficiently modify (and expand) T cells ex vivo provides the opportunity to deliver sufficient immune firepower where it has heretofore been lacking. Sustained transgene expression, coupled with enhanced in vivo engraftment capability, will move adoptive immunotherapy into a realm where longterm therapeutic benefits are the norm rather than the exception.
Genetic Modification of T Lymphocytes for Adoptive Immunotherapy

Claudia Rossig1 and Malcolm K. Brenner2
Molecular Therapy (2004) 10, 5–18;   http://dx.doi.org:/10.1016/j.ymthe.2004.04.014      http://www.nature.com/mt/journal/v10/n1/full/mt20041193a.html

Adoptive transfer of T lymphocytes is a promising therapy for malignancies—particularly of the hemopoietic system—and for otherwise intractable viral diseases. Efforts to broaden the approach have been limited by the physiology of the T cells themselves and by a range of immune evasion mechanisms developed by tumor cells. In this review we show how genetic modification of T cells is being used preclinically and in patients to overcome these limitations, by incorporation of novel receptors, resistance mechanisms, and control genes. We also discuss how the increasing safety and effectiveness of gene transfer technologies will lead to an increase in the use of gene-modified T cells for the treatment of a wider range of disorders.

That gene transfer could be used to improve the effectiveness of T lymphocytes was apparent from the beginning of clinical studies in the field. T cells were the very first targets for genetic modification in human gene transfer experiments. Rosenberg’s group marked tumor-infiltrating lymphocytes ex vivo with a Moloney retroviral vector encoding neomycin phosphotransferase before reinfusing them and attempting to demonstrate selective accumulation at tumor sites. Shortly thereafter, Blaese and Anderson led a group that infused corrected T cells into two children with severe combined immunodeficiency due to ADA deficiency. While neither study was completely successful in terms of outcome, both showed the feasibility of ex vivo gene transfer into human cells and set the stage for many of the studies that followed. More recently, a second wave of interest in adoptive T cell therapies has developed, based on their success in the prevention and treatment of viral infections such as EBV and cytomegalovirus (CMV) and on their apparent ability to eradicate hematologic and perhaps solid malignancies1,2,3,4,5,6. There has been a corresponding increase in studies directed toward enhancing the antineoplastic and antiviral properties of the T cells. In this article we will review how gene transfer may be used to produce the desired improvements focusing on vectors and genes that have had clinical application.

Currently available viral and nonviral vector systems lack a pattern of biodistribution that would favor T cell transduction in vivo—as occurs, for example, with adenovectors and the liver or liposomal vectors and the lung. This lack of favorable biodistribution cannot yet be compensated for by the introduction of specific T-cell-targeting ligands into vectors. Hence, all T cell gene transfer studies conducted to date have used ex vivo transduction followed by adoptive transfer of gene-modified cells. This approach is inherently less attractive for commercial development than directin vivo gene transfer and has probably restricted interest in developing clinical applications using these cells. On the other hand, ex vivo transduction may be more readily controlled, characterized, and standardized than in vivo efforts and may ultimately produce a better defined final product (the transduced cell).

The gene products of suicide and coexpressed resistance genes are highly immunogenic and may induce immune-mediated rejection of the transduced cells. In one study, the persistence of adoptively transferred autologous CD8+ HIV-specific CTL clones modified to express the hygromycin phosphotransferase (Hy) gene and the herpesvirus thymidine kinase gene as a fusion gene was limited by the induction of a potent CD8+ class I MHC-restricted CTL response specific for epitopes derived from the Hy-tk protein126. Less immunogenic suicide and selection marker genes, preferably of human origin, may reduce the immunological inactivation of genetically modified donor lymphocytes. Human-derived prodrug-activating systems include the human folylpolyglutamate synthetase/methotrexate127, the deoxycytidine/cytosine arabinoside128, or the carboxylesterase/irinotecan129 systems. These systems do not activate nontoxic prodrugs but are based on enhancement of already potent chemotherapeutic agents. The administration of methotrexate to treat severe GVHD may not only kill transduced donor lymphocytes but may also have additional inhibitory activity on nontransduced but activated T cells.

Finally, endogenous proapoptotic molecules have been proposed as nonimmunogenic suicide genes. A chimeric protein that contains the FK506-binding protein FKBP12 linked to the intracellular domain of human Fas130 was recently introduced. Addition of the dimerizing prodrug induces Fas crosslinking with subsequent triggering of an apoptotic death signal.

Genetic engineering of T lymphocytes should help deliver on the promise of immunotherapies for cancer, infection, and autoimmune disease. Improvements in transduction, selection, and expansion techniques and the development of new viral vectors incapable of insertional mutagenesis will reduce the risks and further enhance the integration of T cell and gene therapies. Nonetheless, successful application of the proposed modifications to the clinical setting still requires many iterative studies to allow investigators to optimize the individual components of the approach.

Genetically modified T cells in cancer therapy: opportunities and challenges
Michaela Sharpe, Natalie Mount

 

The feasibility of T-cell adoptive transfer was first reported nearly 20 years ago (Walter et al., 1995) and the field of T-cell therapies is now poised for significant clinical advances. Recent clinical trial successes have been achieved through multiple small advances, improved understanding of immunology and emerging technologies. As the key challenges of T-cell avidity, persistence and ability to exert the desired anti-tumour effects as well as the identification of new target antigens are addressed, a broader clinical application of these therapies could be achieved. As the clinical data emerges, the challenge of making these therapies available to patients shifts to implementing robust, scalable and cost-effective manufacture and to the further evolution of the regulatory requirements to ensure an appropriate but proportionate system that is adapted to the characteristics of these innovative new medicines.

 

 

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