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Regulatory T cells (Tregs) are important for sperm tolerance and male fertility
Reporter and Curator: Dr. Sudipta Saha, Ph.D.
Regulatory T cells (Tregs) are specialized immune cells that modulate tissue homeostasis. They are a specialized subset of T lymphocytes that function as suppressive immune cells and inhibit various elements of immune response in vitro and in vivo. While there are constraints on the number or function of Tregs which can be exploited to evoke an effective anti-tumor response, sufficient expansion of Tregs is essential for successful organ transplantation and for promoting tolerance of self and foreign antigens. Current studies have provided evidence that a defect in the number or function of Tregs contributes to the etiology of several reproductive diseases.
In the male reproductive tract, prevention of autoimmune responses against antigenic spermatozoa, while ensuring protection against stressors, is a key determinant of fertility. Using an autoimmunity-induced model, it was uncovered that the role of Tregs in maintaining the tolerogenic state of the testis and epididymis. The loss of tolerance induced an exacerbated immune cell infiltration and the development of anti-sperm antibodies, which caused severe male subfertility. By identifying immunoregulatory mechanisms in the testis and epididymis.
Tregs modulate tissue homeostatic processes and immune responses. Understanding tissue-Treg biology will contribute to developing precision-targeting treatment strategies. Here, it was reported that Tregs maintain the tolerogenic state of the testis and epididymis, where sperm are produced and mature. It was found that Treg depletion induces severe autoimmune orchitis and epididymitis, manifested by an exacerbated immune cell infiltration [CD4 T cells, monocytes, and mononuclear phagocytes (MPs)] and the development of anti-sperm antibodies (ASA).
In Treg-depleted mice, MPs increased projections toward the epididymal lumen as well as invading the lumen. ASA-bound sperm enhance sperm agglutination and might facilitate sperm phagocytosis. Tolerance breakdown impaired epididymal epithelial function and altered extracellular vesicle cargo, both of which play crucial roles in the acquisition of sperm fertilizing ability and subsequent embryo development. The affected mice had reduced sperm number and motility and severe fertility defects.
Deciphering these immunoregulatory mechanisms may lead to the development of therapies for infertility and identifying potential targets for immuno-contraception. Ultimately, such knowledge fills gaps related to reproductive mucosa, which is an understudied facet of human male health.
Eight Subcellular Pathologies driving Chronic Metabolic Diseases – Methods for Mapping Bioelectronic Adjustable Measurements as potential new Therapeutics: Impact on Pharmaceuticals in Use
In this curation we wish to present two breaking through goals:
Goal 1:
Exposition of a new direction of research leading to a more comprehensive understanding of Metabolic Dysfunctional Diseases that are implicated in effecting the emergence of the two leading causes of human mortality in the World in 2023: (a) Cardiovascular Diseases, and (b) Cancer
Goal 2:
Development of Methods for Mapping Bioelectronic Adjustable Measurements as potential new Therapeutics for these eight subcellular causes of chronic metabolic diseases. It is anticipated that it will have a potential impact on the future of Pharmaceuticals to be used, a change from the present time current treatment protocols for Metabolic Dysfunctional Diseases.
According to Dr. Robert Lustig, M.D, an American pediatric endocrinologist. He is Professor emeritus of Pediatrics in the Division of Endocrinology at the University of California, San Francisco, where he specialized in neuroendocrinology and childhood obesity, there are eight subcellular pathologies that drive chronic metabolic diseases.
These eight subcellular pathologies can’t be measured at present time.
In this curation we will attempt to explore methods of measurement for each of these eight pathologies by harnessing the promise of the emerging field known as Bioelectronics.
Unmeasurable eight subcellular pathologies that drive chronic metabolic diseases
Glycation
Oxidative Stress
Mitochondrial dysfunction [beta-oxidation Ac CoA malonyl fatty acid]
Insulin resistance/sensitive [more important than BMI], known as a driver to cancer development
Membrane instability
Inflammation in the gut [mucin layer and tight junctions]
Epigenetics/Methylation
Autophagy [AMPKbeta1 improvement in health span]
Diseases that are not Diseases: no drugs for them, only diet modification will help
Image source
Robert Lustig, M.D. on the Subcellular Processes That Belie Chronic Disease
These eight Subcellular Pathologies driving Chronic Metabolic Diseases are becoming our focus for exploration of the promise of Bioelectronics for two pursuits:
Will Bioelectronics be deemed helpful in measurement of each of the eight pathological processes that underlie and that drive the chronic metabolic syndrome(s) and disease(s)?
IF we will be able to suggest new measurements to currently unmeasurable health harming processes THEN we will attempt to conceptualize new therapeutic targets and new modalities for therapeutics delivery – WE ARE HOPEFUL
In the Bioelecronics domain we are inspired by the work of the following three research sources:
Michael Levin is an American developmental and synthetic biologist at Tufts University, where he is the Vannevar Bush Distinguished Professor. Levin is a director of the Allen Discovery Center at Tufts University and Tufts Center for Regenerative and Developmental Biology. Wikipedia
THE VOICE of Dr. Justin D. Pearlman, MD, PhD, FACC
PENDING
THE VOICE of Stephen J. Williams, PhD
Ten TakeAway Points of Dr. Lustig’s talk on role of diet on the incidence of Type II Diabetes
25% of US children have fatty liver
Type II diabetes can be manifested from fatty live with 151 million people worldwide affected moving up to 568 million in 7 years
A common myth is diabetes due to overweight condition driving the metabolic disease
There is a trend of ‘lean’ diabetes or diabetes in lean people, therefore body mass index not a reliable biomarker for risk for diabetes
Thirty percent of ‘obese’ people just have high subcutaneous fat. the visceral fat is more problematic
there are people who are ‘fat’ but insulin sensitive while have growth hormone receptor defects. Points to other issues related to metabolic state other than insulin and potentially the insulin like growth factors
At any BMI some patients are insulin sensitive while some resistant
Visceral fat accumulation may be more due to chronic stress condition
Fructose can decrease liver mitochondrial function
A methionine and choline deficient diet can lead to rapid NASH development
New studies link cell cycle proteins to immunosurveillance of premalignant cells
Curator: Stephen J. Williams, Ph.D.
The following is from a Perspectives article in the journal Science by Virinder Reen and Jesus Gil called “Clearing Stressed Cells: Cell cycle arrest produces a p21-dependent secretome that initaites immunosurveillance of premalignant cells”. This is a synopsis of the Sturmlechener et al. research article in the same issue (2).
Complex organisms repair stress-induced damage to limit the replication of faulty cells that could drive cancer. When repair is not possible, tissue homeostasis is maintained by the activation of stress response programs such as apoptosis, which eliminates the cells, or senescence, which arrests them (1). Cellular senescence causes the arrest of damaged cells through the induction of cyclin-dependent kinase inhibitors (CDKIs) such as p16 and p21 (2). Senescent cells also produce a bioactive secretome (the senescence-associated secretory phenotype, SASP) that places cells under immunosurveillance, which is key to avoiding the detrimental inflammatory effects caused by lingering senescent cells on surrounding tissues. On page 577 of this issue, Sturmlechner et al. (3) report that induction of p21 not only contributes to the arrest of senescent cells, but is also an early signal that primes stressed cells for immunosurveillance.Senescence is a complex program that is tightly regulated at the epigenetic and transcriptional levels. For example, exit from the cell cycle is controlled by the induction of p16 and p21, which inhibit phosphorylation of the retinoblastoma protein (RB), a transcriptional regulator and tumor suppressor. Hypophosphorylated RB represses transcription of E2F target genes, which are necessary for cell cycle progression. Conversely, production of the SASP is regulated by a complex program that involves super-enhancer (SE) remodeling and activation of transcriptional regulators such as nuclear factor κB (NF-κB) or CCAAT enhancer binding protein–β (C/EBPβ) (4).
Senescence is a complex program that is tightly regulated at the epigenetic and transcriptional levels. For example, exit from the cell cycle is controlled by the induction of p16 and p21, which inhibit phosphorylation of the retinoblastoma protein (RB), a transcriptional regulator and tumor suppressor. Hypophosphorylated RB represses transcription of E2F target genes, which are necessary for cell cycle progression. Conversely, production of the SASP is regulated by a complex program that involves super-enhancer (SE) remodeling and activation of transcriptional regulators such as nuclear factor κB (NF-κB) or CCAAT enhancer binding protein–β (C/EBPβ) (4).
Sturmlechner et al. found that activation of p21 following stress rapidly halted cell cycle progression and triggered an internal biological timer (of ∼4 days in hepatocytes), allowing time to repair and resolve damage (see the figure). In parallel, C-X-C motif chemokine 14 (CXCL14), a component of the PASP, attracted macrophages to surround and closely surveil these damaged cells. Stressed cells that recovered and normalized p21 expression suspended PASP production and circumvented immunosurveillance. However, if the p21-induced stress was unmanageable, the repair timer expired, and the immune cells transitioned from surveillance to clearance mode. Adjacent macrophages mounted a cytotoxic T lymphocyte response that destroyed damaged cells. Notably, the overexpression of p21 alone was sufficient to orchestrate immune killing of stressed cells, without the need of a senescence phenotype. Overexpression of other CDKIs, such as p16 and p27, did not trigger immunosurveillance, likely because they do not induce CXCL14 expression.In the context of cancer, senescent cell clearance was first observed following reactivation of the tumor suppressor p53 in liver cancer cells. Restoring p53 signaling induced senescence and triggered the elimination of senescent cells by the innate immune system, prompting tumor regression (5). Subsequent work has revealed that the SASP alerts the immune system to target preneoplastic senescent cells. Hepatocytes expressing the oncogenic mutant NRASG12V (Gly12→Val) become senescent and secrete chemokines and cytokines that trigger CD4+ T cell–mediated clearance (6). Despite the relevance for tumor suppression, relatively little is known about how immunosurveillance of oncogene-induced senescent cells is initiated and controlled.
Source of image: Reen, V. and Gil, J. Clearing Stressed Cells. Science Perspectives 2021;Vol 374(6567) p 534-535.
References
2. Sturmlechner I, Zhang C, Sine CC, van Deursen EJ, Jeganathan KB, Hamada N, Grasic J, Friedman D, Stutchman JT, Can I, Hamada M, Lim DY, Lee JH, Ordog T, Laberge RM, Shapiro V, Baker DJ, Li H, van Deursen JM. p21 produces a bioactive secretome that places stressed cells under immunosurveillance. Science. 2021 Oct 29;374(6567):eabb3420. doi: 10.1126/science.abb3420. Epub 2021 Oct 29. PMID: 34709885.
More Articles on Cancer, Senescence and the Immune System in this Open Access Online Scientific Journal Include
Yet another Success Story: Machine Learning to predict immunotherapy response
Curator and Reporter: Dr. Premalata Pati, Ph.D., Postdoc
Immune-checkpoint blockers(ICBs) immunotherapy appears promising for various cancer types, offering a durable therapeutic advantage. Only a number of cases with cancer respond to this therapy. Biomarkers are required to adequately predict the responses of patients. This article evaluates this issue utilizing a system method to characterize the immune response of the anti-tumor based on the entire tumor environment. Researchers build mechanical biomarkers and cancer-specific response models using interpretable machine learning that predict the response of patients to ICB.
The lymphatic and immunological systems help the body defend itself by combating. The immune system functions as the body’s own personal police force, hunting down and eliminating pathogenic baddies.
According to Federica Eduati, Department of Biomedical Engineering at TU/e, “The immune system of the body is quite adept at detecting abnormally behaving cells. Cells that potentially grow into tumors or cancer in the future are included in this category. Once identified, the immune system attacks and destroys the cells.”
Immunotherapy and machine learning are combining to assist the immune system solve one of its most vexing problems: detecting hidden tumorous cells in the human body.
It is the fundamental responsibility of our immune system to identify and remove alien invaders like bacteria or viruses, but also to identify risks within the body, such as cancer. However, cancer cells have sophisticated ways of escaping death by shutting off immune cells. Immunotherapy can reverse the process, but not for all patients and types of cancer. To unravel the mystery, Eindhoven University of Technology researchers used machine learning. They developed a model to predict whether immunotherapy will be effective for a patient using a simple trick. Even better, the model outperforms conventional clinical approaches.
“Tumor also contains multiple types of immune and fibroblast cells which can play a role in favor of or anti-tumor, and communicates among themselves,” said Oscar Lapuente-Santana, a researcher doctoral student in the computational biology group. “We had to learn how complicated regulatory mechanisms in the micro-environment of the tumor affect the ICB response. We have used RNA sequencing datasets to depict numerous components of the Tumor Microenvironment (TME) in a high-level illustration.”
Using computational algorithms and datasets from previous clinical patient care, the researchers investigated the TME.
Eduati explained
While RNA-sequencing databases are publically available, information on which patients responded to ICB therapy is only available for a limited group of patients and cancer types. So, to tackle the data problem, we used a trick.
All 100 models learned in the randomized cross-validation were included in the EaSIeR tool. For each validation dataset, we used the corresponding cancer-type-specific model: SKCM for the melanoma Gide, Auslander, Riaz, and Liu cohorts; STAD for the gastric cancer Kim cohort; BLCA for the bladder cancer Mariathasan cohort; and GBM for the glioblastoma Cloughesy cohort. To make predictions for each job, the average of the 100 cancer-type-specific models was employed. The predictions of each dataset’s cancer-type-specific models were also compared to models generated for the remaining 17 cancer types.
From the same datasets, the researchers selected several surrogate immunological responses to be used as a measure of ICB effectiveness.
Lapuente-Santana stated
One of the most difficult aspects of our job was properly training the machine learning models. We were able to fix this by looking at alternative immune responses during the training process.
DREAM is an organization that carries out crowd-based tasks with biomedical algorithms. “We were the first to compete in one of the sub-challenges under the name cSysImmunoOnco team,” Eduati remarks.
The researchers noted,
We applied machine learning to seek for connections between the obtained system-based attributes and the immune response, estimated using 14 predictors (proxies) derived from previous publications. We treated these proxies as individual tasks to be predicted by our machine learning models, and we employed multi-task learning algorithms to jointly learn all tasks.
The researchers discovered that their machine learning model surpasses biomarkers that are already utilized in clinical settings to evaluate ICB therapies.
But why are Eduati, Lapuente-Santana, and their colleagues using mathematical models to tackle a medical treatment problem? Is this going to take the place of the doctor?
Eduati explains
Mathematical models can provide an overview of the interconnection between individual molecules and cells and at the same time predicting a particular patient’s tumor behavior. This implies that immunotherapy with ICB can be personalized in a patient’s clinical setting. The models can aid physicians with their decisions about optimum therapy, it is vital to note that they will not replace them.
Furthermore, the model aids in determining which biological mechanisms are relevant for the biological response.
The researchers noted
Another advantage of our concept is that it does not need a dataset with known patient responses to immunotherapy for model training.
Further testing is required before these findings may be implemented in clinical settings.
Main Source:
Lapuente-Santana, Ó., van Genderen, M., Hilbers, P. A., Finotello, F., & Eduati, F. (2021). Interpretable systems biomarkers predict response to immune-checkpoint inhibitors. Patterns, 100293. https://www.cell.com/patterns/pdfExtended/S2666-3899(21)00126-4
Other Related Articles published in this Open Access Online Scientific Journal include the following:
Inhibitory CD161 receptor recognized as a potential immunotherapy target in glioma-infiltrating T cells by single-cell analysis
Deep Learning for In-silico Drug Discovery and Drug Repurposing: Artificial Intelligence to search for molecules boosting response rates in Cancer Immunotherapy: Insilico Medicine @John Hopkins University
Inhibitory CD161 receptor recognized as a potential immunotherapy target in glioma-infiltrating T cells by single-cell analysis
Reporter: Dr. Premalata Pati, Ph.D., Postdoc
Brain tumors, especially the diffused Gliomas are of the most devastating forms of cancer and have so-far been resistant to immunotherapy. It is comprehended that T cells can penetrate the glioma cells, but it still remains unknown why infiltrating cells miscarry to mount a resistant reaction or stop the tumor development.
Gliomas are brain tumors that begin from neuroglial begetter cells. The conventional therapeutic methods including, surgery, chemotherapy, and radiotherapy, have accomplished restricted changes inside glioma patients. Immunotherapy, a compliance in cancer treatment, has introduced a promising strategy with the capacity to penetrate the blood-brain barrier. This has been recognized since the spearheading revelation of lymphatics within the central nervous system. Glioma is not generally carcinogenic. As observed in a number of cases, the tumor cells viably reproduce and assault the adjoining tissues, by and large, gliomas are malignant in nature and tend to metastasize. There are four grades in glioma, and each grade has distinctive cell features and different treatment strategies. Glioblastoma is a grade IV glioma, which is the crucial aggravated form. This infers that all glioblastomas are gliomas, however, not all gliomas are glioblastomas.
Decades of investigations on infiltrating gliomas still take off vital questions with respect to the etiology, cellular lineage, and function of various cell types inside glial malignancies. In spite of the available treatment options such as surgical resection, radiotherapy, and chemotherapy, the average survival rate for high-grade glioma patients remains 1–3 years (1).
A recent in vitro study performed by the researchers of Dana-Farber Cancer Institute, Massachusetts General Hospital, and the Broad Institute of MIT and Harvard, USA, has recognized that CD161 is identified as a potential new target for immunotherapy of malignant brain tumors. The scientific team depicted their work in the Cell Journal, in a paper entitled, “Inhibitory CD161 receptor recognized in glioma-infiltrating T cells by single-cell analysis.” on 15th February 2021.
To further expand their research and findings, Dr. Kai Wucherpfennig, MD, PhD, Chief of the Center for Cancer Immunotherapy, at Dana-Farber stated that their research is additionally important in a number of other major human cancer types such as
melanoma,
lung,
colon, and
liver cancer.
Dr. Wucherpfennig has praised the other authors of the report Mario Suva, MD, PhD, of Massachusetts Common Clinic; Aviv Regev, PhD, of the Klarman Cell Observatory at Broad Institute of MIT and Harvard, and David Reardon, MD, clinical executive of the Center for Neuro-Oncology at Dana-Farber.
Hence, this new study elaborates the effectiveness of the potential effectors of anti-tumor immunity in subsets of T cells that co-express cytotoxic programs and several natural killer (NK) cell genes.
The Study-
IMAGE SOURCE: Experimental Strategy (Mathewson et al., 2021)
The group utilized single-cell RNA sequencing (RNA-seq) to mull over gene expression and the clonal picture of tumor-infiltrating T cells. It involved the participation of 31 patients suffering from diffused gliomas and glioblastoma. Their work illustrated that the ligand molecule CLEC2D activates CD161, which is an immune cell surface receptor that restrains the development of cancer combating activity of immune T cells and tumor cells in the brain. The study reveals that the activation of CD161 weakens the T cell response against tumor cells.
Based on the study, the facts suggest that the analysis of clonally expanded tumor-infiltrating T cells further identifies the NK gene KLRB1 that codes for CD161 as a candidate inhibitory receptor. This was followed by the use of
CRISPR/Cas9 gene-editing technology to inactivate the KLRB1 gene in T cells and showed that CD161 inhibits the tumor cell-killing function of T cells. Accordingly,
genetic inactivation of KLRB1 or
antibody-mediated CD161 blockade
enhances T cell-mediated killing of glioma cells in vitro and their anti-tumor function in vivo. KLRB1 and its associated transcriptional program are also expressed by substantial T cell populations in other forms of human cancers. The work provides an atlas of T cells in gliomas and highlights CD161 and other NK cell receptors as immune checkpoint targets.
Further, it has been identified that many cancer patients are being treated with immunotherapy drugs that disable their “immune checkpoints” and their molecular brakes are exploited by the cancer cells to suppress the body’s defensive response induced by T cells against tumors. Disabling these checkpoints lead the immune system to attack the cancer cells. One of the most frequently targeted checkpoints is PD-1. However, recent trials of drugs that target PD-1 in glioblastomas have failed to benefit the patients.
In the current study, the researchers found that fewer T cells from gliomas contained PD-1 than CD161. As a result, they said, “CD161 may represent an attractive target, as it is a cell surface molecule expressed by both CD8 and CD4 T cell subsets [the two types of T cells engaged in response against tumor cells] and a larger fraction of T cells express CD161 than the PD-1 protein.”
However, potential side effects of antibody-mediated blockade of the CLEC2D-CD161 pathway remain unknown and will need to be examined in a non-human primate model. The group hopes to use this finding in their future work by
utilizing their outline by expression of KLRB1 gene in tumor-infiltrating T cells in diffuse gliomas to make a remarkable contribution in therapeutics related to immunosuppression in brain tumors along with four other common human cancers ( Viz. melanoma, non-small cell lung cancer (NSCLC), hepatocellular carcinoma, and colorectal cancer) and how this may be manipulated for prevalent survival of the patients.
References
(1) Anders I. Persson, QiWen Fan, Joanna J. Phillips, William A. Weiss, 39 – Glioma, Editor(s): Sid Gilman, Neurobiology of Disease, Academic Press, 2007, Pages 433-444, ISBN 9780120885923, https://doi.org/10.1016/B978-012088592-3/50041-4.
4.1.3 Single-cell Genomics: Directions in Computational and Systems Biology – Contributions of Prof. Aviv Regev @Broad Institute of MIT and Harvard, Cochair, the Human Cell Atlas Organizing Committee with Sarah Teichmann of the Wellcome Trust Sanger Institute
4.1.7 Norwich Single-Cell Symposium 2019, Earlham Institute, single-cell genomics technologies and their application in microbial, plant, animal and human health and disease, October 16-17, 2019, 10AM-5PM
Positron Emission Tomography (PET) and Near-Infrared Fluorescence Imaging: Noninvasive Imaging of Cancer Stem Cells (CSCs) monitoring of AC133+ glioblastoma in subcutaneous and intracerebral xenograft tumors
LPBI Group’s decision to publish the Table of Contents of this Report does not imply endorsement of the Report
Aviva Lev-Ari, PhD, RN, Founder 1.0 & 2.0 LPBI Group
Guest Reporter: MIKE WOOD
Marketing Executive BIOTECH FORECASTS
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CAR T-cell therapy as a part of adoptive cell therapy (ACT), has become one of the most rapidly growing and promising fields in the Immuno-oncology. As compared to the conventional cancer therapies, CAR T-cell therapy is the single-dose solution for the treatment of various cancers, significantly for some lethal forms of hematological malignancies.
CAR T-cell therapy mainly involves the use of engineered T-cells, the process starts with the extraction of T-cells through leukapheresis, either from the patient (autologous) or a healthy donor (allogeneic). After the expression of a synthetic receptor (Chimeric Antigen Receptor) in the lab, the altered T-cells are expanded to the right dose and administered into the patient’s body. where they target and attach to a specific antigen on the tumor surface, to kill the cancerous cells by igniting the apoptosis.
The global CAR T-cell therapy market was valued at $734 million in 2019 and is estimated to reach $4,078 million by 2027, registering a CAGR of 23.91% from 2020 to 2027.
Factors that drive the market growth involve, (1)Increased in fundingfor R&D activities pertaining to cell and gene therapy. By H1 2020 cell and gene therapy companies set new records in the fundraising despite the pandemic crisis. For Instance, by June 2020 totaled $1,452 Million raised in Five IPOs including, Legend Biotech ($487M), Passage Bio ($284M), Akouos ($244M), Generation Bio ($230M), and Beam Therapeutics ($207M), which is 2.5 times the total IPO of 2019.
Moreover, in 2019 cell therapy companies specifically have raised $560 million of venture capital, including Century Therapeutics ($250M), Achilles Therapeutics Ltd. ($121M in series B), NKarta Therapeutics Inc. ($114M), and Tmunity Therapeutics ($75M in Series B).
(2)Increased in No. of Approved Products, By July 2020, there are a total of 03 approved CAR T-cell therapy products, including KYMRIAH®, YESCARTA®, and the most recently approved TECARTUS™ (formerly KTE-X19). Furthermore, two CAR T-cell therapies BB2121, and JCAR017 are expected to get the market approval by the end of 2020 or in early 2021.
Other factors that boost the market growth involves; (3) increase in government support, (4) ethical acceptance of Cell and Gene therapy for cancer treatment, (5) rise in the prevalence of cancer, and (6) an increase in awareness regarding the CAR T-cell therapy.
However, high costs associated with the treatment (KYMRIAH® cost around $475,000, and YESCARTA® costs $373,000 per infusion), long production hours, obstacles in treating solid tumors, and unwanted immune responses & potential side effects might hamper the market growth.
The report also presents a detailed quantitative analysis of the current market trends and future estimations from 2020 to 2027.
The forecasts cover 2 Approach Types, 5 Antigen Types, 5 Application Types, 4 Regions, and 14 Countries.
The report comes with an associated file covering quantitative data from all numeric forecasts presented in the report, as well as with a Clinical Trials Data File.
KEY FINDINGS
The report has the following key findings:
The global CAR T-cell therapy market accounted for $734 million in 2019 and is estimated to reach $4,078 million by 2027, registering a CAGR of 23.91% from 2020 to 2027.
By approach type the autologous segment was valued at $655.26 million in 2019 and is estimated to reach $ 3,324.52 million by 2027, registering a CAGR of 22.51% from 2020 to 2027.
By approach type, the allogeneic segment exhibits the highest CAGR of 32.63%.
Based on the Antigen segment CD19 was the largest contributor among the other segments in 2019.
The Acute lymphocytic leukemia (ALL) segment generated the highest revenue and is expected to continue its dominance in the future, followed by the Diffuse large B-cell lymphoma (DLBCL) segment.
North America dominated the global CAR T-cell therapy market in 2019 and is projected to continue its dominance in the future.
China is expected to grow the highest in the Asia-Pacific region during the forecast period.
TOPICS COVERED
The report covers the following topics:
Market Drivers, Restraints, and Opportunities
Porters Five Forces Analysis
CAR T-Cell Structure, Generations, Manufacturing, and Pricing Models
Top Winning Strategies, Top Investment Pockets
Analysis of by Approach Type, Antigen Type, Application, and Region
51 Company Profiles, Product Portfolio, and Key Strategies
Approved Products Profiles, and list of Expected Approvals
COVID-19 Impact on the Cell and Gene Therapy Industry
CAR T-cell therapy clinical trials analysis from 1997 to 2019
Market analysis and forecasts from 2020 to 2027
FORECAST SEGMENTATION
By Approach Type
Autologous
Allogeneic
By Antigen Type
CD19
CD20
BCMA
MSLN
Others
By Application
Acute lymphoblastic leukemia (ALL)
Diffuse large B-Cell lymphoma (DLBCL)
Multiple Myeloma (MM)
Acute Myeloid Leukemia (AML)
Other Cancer Indications
By Region
North America: USA, Canada, Mexico
Europe: UK, Germany, France, Spain, Italy, Rest of Europe
Asia-Pacific: China, Japan, India, South Korea, Rest of Asia-Pacific
LAMEA: Brazil, South Africa, Rest of LAMEA
Contact at info@biotechforecasts.com for any Queries or Free Report Sample
there have been many instances of off-target effects where genes, other than the selected target, are edited out. This ‘off-target’ issue has hampered much of the utility of CRISPR in gene-therapy and CART therapy
However, an article in Science by Jon Cohen explains a Nature paper’s finding of a new tool in the CRISPR arsenal called prime editing, meant to increase CRISPR specificity and precision editing capabilities.
Primeediting promises to be a cut above CRISPR Jon Cohen CRISPR, an extraordinarily powerful genome-editing tool invented in 2012, can still be clumsy. … Primeediting steers around shortcomings of both techniques by heavily modifying the Cas9 protein and the guide RNA. … ” Primeediting “well may become the way that disease-causing mutations are repaired,” he says.
The effort, led by Drs. David Liu and Andrew Anzalone at the Broad Institute (Cambridge, MA), relies on the modification of the Cas9 protein and guide RNA, so that there is only a nick in a single strand of the double helix. The canonical Cas9 cuts both strands of DNA, and so relies on an efficient gap repair activity of the cell. The second part, a new type of guide RNA called a pegRNA, contains an RNA template for a new DNA sequence to be added at the target location. This pegRNA-directed synthesis of the new template requires the attachment of a reverse transcriptase enzymes to the Cas9. So far Liu and his colleagues have tested the technology on over 175 human and rodent cell lines with great success. In addition, they had also corrected mutations which cause Tay Sachs disease, which previous CRISPR systems could not do. Liu claims that this technology could correct over 89% of pathogenic variants in human diseases.
A company Prime Medicine has been formed out of this effort.
As was announced, prime editing for human therapeutics will be jointly developed by both Prime Medicine and Beam Therapeutics, each focusing on different types of edits and distinct disease targets, which will help avoid redundancy and allow us to cover more disease territory overall. The companies will also share knowledge in prime editing as well as in accompanying technologies, such as delivery and manufacturing.
Reader of StatNews.: Can you please compare the pros and cons of prime editing versus base editing?
The first difference between base editing and prime editing is that base editing has been widely used for the past 3 1/2 years in organisms ranging from bacteria to plants to mice to primates. Addgene tells me that the DNA blueprints for base editors from our laboratory have been distributed more than 7,500 times to more than 1,000 researchers around the world, and more than 100 research papers from many different laboratories have been published using base editors to achieve desired gene edits for a wide variety of applications. While we are very excited about prime editing, it’s brand-new and there has only been one paper published thus far. So there’s much to do before we can know if prime editing will prove to be as general and robust as base editing has proven to be.
We directly compared prime editors and base editors in our study, and found that current base editors can offer higher editing efficiency and fewer indel byproducts than prime editors, while prime editors offer more targeting flexibility and greater editing precision. So when the desired edit is a transition point mutation (C to T, T to C, A to G, or G to A), and the target base is well-positioned for base editing (that is, a PAM sequence exists approximately 15 bases from the target site), then base editing can result in higher editing efficiencies and fewer byproducts. When the target base is not well-positioned for base editing, or when other “bystander” C or A bases are nearby that must not be edited, then prime editing offers major advantages since it does not require a precisely positioned PAM sequence and is a true “search-and-replace” editing capability, with no possibility of unwanted bystander editing at neighboring bases.
Of course, for classes of mutations other than the four types of point mutations that base editors can make, such as insertions, deletions, and the eight other kinds of point mutations, to our knowledge prime editing is currently the only approach that can make these mutations in human cells without requiring double-stranded DNA cuts or separate DNA templates.
Nucleases (such as the zinc-finger nucleases, TALE nucleases, and the original CRISPR-Cas9), base editors, and prime editors each have complementary strengths and weaknesses, just as scissors, pencils, and word processors each have unique and useful roles. All three classes of editing agents already have or will have roles in basic research and in applications such as human therapeutics and agriculture.
Most genetic variants that contribute to disease1 are challenging to correct efficiently and without excess byproducts2,3,4,5. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few byproducts, the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay–Sachs disease (requiring a deletion in HEXA); to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases.
From Anzolone et al. Nature 2019 Figure 1.
Prime editing strategy
Cas9 targets DNA using a guide RNA containing a spacer sequence that hybridizes to the target DNA site. We envisioned the generation of guide RNAs that both specify the DNA target and contain new genetic information that replaces target DNA nucleotides. To transfer information from these engineered guide RNAs to target DNA, we proposed that genomic DNA, nicked at the target site to expose a 3′-hydroxyl group, could be used to prime the reverse transcription of an edit-encoding extension on the engineered guide RNA (the pegRNA) directly into the target site (Fig. 1b, c, Supplementary Discussion).
These initial steps result in a branched intermediate with two redundant single-stranded DNA flaps: a 5′ flap that contains the unedited DNA sequence and a 3′ flap that contains the edited sequence copied from the pegRNA (Fig. 1c). Although hybridization of the perfectly complementary 5′ flap to the unedited strand is likely to be thermodynamically favoured, 5′ flaps are the preferred substrate for structure-specific endonucleases such as FEN122, which excises 5′ flaps generated during lagging-strand DNA synthesis and long-patch base excision repair. The redundant unedited DNA may also be removed by 5′ exonucleases such as EXO123.
The authors reasoned that preferential 5′ flap excision and 3′ flap ligation could drive the incorporation of the edited DNA strand, creating heteroduplex DNA containing one edited strand and one unedited strand (Fig. 1c).
DNA repair to resolve the heteroduplex by copying the information in the edited strand to the complementary strand would permanently install the edit (Fig. 1c).
They had hypothesized that nicking the non-edited DNA strand might bias DNA repair to preferentially replace the non-edited strand.
Results
The authors evaluated the eukaryotic cell DNA repair outcomes of 3′ flaps produced by pegRNA-programmed reverse transcription in vitro, and performed in vitro prime editing on reporter plasmids, then transformed the reaction products into yeast cells (Extended Data Fig. 2).
Reporter plasmids encoding EGFP and mCherry separated by a linker containing an in-frame stop codon, +1 frameshift, or −1 frameshift were constructed and when plasmids were edited in vitro with Cas9 nickase, RT, and 3′-extended pegRNAs encoding a transversion that corrects the premature stop codon, 37% of yeast transformants expressed both GFP and mCherry (Fig. 1f, Extended Data Fig. 2).
They fused a variant of M—MLV-RT (reverse transcriptase) to Cas9 with an extended linker and this M-MLV RT fused to the C terminus of Cas9(H840A) nickase was designated as PE1. This strategy allowed the authors to generate a cell line containing all the required components of the primer editing system. They constructed 19 variants of PE1 containing a variety of RT mutations to evaluate their editing efficiency in human cells
Generated a pentamutant RT incorporated into PE1 (Cas9(H840A)–M-MLV RT(D200N/L603W/T330P/T306K/W313F)) is hereafter referred to as prime editor 2 (PE2). These were more thermostable versions of RT with higher efficiency.
Optimized the guide (pegRNA) using a series of permutations and recommend starting with about 10–16 nt and testing shorter and longer RT templates during pegRNA optimization.
In the previous attempts (PE1 and PE2 systems), mismatch repair resolves the heteroduplex to give either edited or non-edited products. So they next developed an optimal editing system (PE3) to produce optimal nickase activity and found nicks positioned 3′ of the edit about 40–90 bp from the pegRNA-induced nick generally increased editing efficiency (averaging 41%) without excess indel formation (6.8% average indels for the sgRNA with the highest editing efficiency) (Fig. 3b).
The cell line used to finalize and validate the system was predominantly HEK293T immortalized cell line
Together, their findings establish that PE3 systems improve editing efficiencies about threefold compared with PE2, albeit with a higher range of indels than PE2. When it is possible to nick the non-edited strand with an sgRNA that requires editing before nicking, the PE3b system offers PE3-like editing levels while greatly reducing indel formation.
Off Target Effects: Strikingly, PE3 or PE2 with the same 16 pegRNAs containing these four target spacers resulted in detectable off-target editing at only 3 out of 16 off-target sites, with only 1 of 16 showing an off-target editing efficiency of 1% or more (Extended Data Fig. 6h). Average off-target prime editing for pegRNAs targeting HEK3, HEK4, EMX1, and FANCFat the top four known Cas9 off-target sites for each protospacer was <0.1%, <2.2 ± 5.2%, <0.1%, and <0.13 ± 0.11%, respectively (Extended Data Fig. 6h).
The PE3 system was very efficient at editing the most common mutation that causes Tay-Sachs disease, a 4-bp insertion in HEXA(HEXA1278+TATC).
References
Landrum, M. J. et al. ClinVar: public archive of interpretations of clinically relevant variants. Nucleic Acids Res. 44, D862–D868 (2016).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science337, 816–821 (2012).
Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Biotechnol. 36, 765–771 (2018).
As part of the all-of-America approach to fighting the COVID-19 pandemic, the U.S. Food and Drug Administration has been working with partners across the U.S. government, academia and industry to expedite the development and availability of critical medical products to treat this novel virus. Today, we are providing an update on one potential treatment called convalescent plasma and encouraging those who have recovered from COVID-19 to donate plasma to help others fight this disease.
Convalescent plasma is an antibody-rich product made from blood donated by people who have recovered from the disease caused by the virus. Prior experience with respiratory viruses and limited data that have emerged from China suggest that convalescent plasma has the potential to lessen the severity or shorten the length of illness caused by COVID-19. It is important that we evaluate this potential therapy in the context of clinical trials, through expanded access, as well as facilitate emergency access for individual patients, as appropriate.
The response to the agency’s recently announced national efforts to facilitate the development of and access to convalescent plasma has been tremendous. More than 1,040 sites and 950 physician investigators nationwide have signed on to participate in the Mayo Clinic-ledExternal Link Disclaimer expanded access protocol. A number of clinical trials are also taking place to evaluate the safety and efficacy of convalescent plasma and the FDA has granted numerous single patient emergency investigational new drug (eIND) applications as well.
FDA issues guidelines on clinical trials and obtaining emergency enrollment concerning convalescent plasma
FDA has issued guidance to provide recommendations to health care providers and investigators on the administration and study of investigational convalescent plasma collected from individuals who have recovered from COVID-19 (COVID-19 convalescent plasma) during the public health emergency.
The guidance provides recommendations on the following:
Because COVID-19 convalescent plasma has not yet been approved for use by FDA, it is regulated as an investigational product. A health care provider must participate in one of the pathways described below. FDA does not collect COVID-19 convalescent plasma or provide COVID-19 convalescent plasma. Health care providers or acute care facilities should instead obtain COVID-19 convalescent plasma from an FDA-registered blood establishment.
Excerpts from the guidance document are provided below.
Background
The Food and Drug Administration (FDA or Agency) plays a critical role in protecting the United States (U.S.) from threats including emerging infectious diseases, such as the Coronavirus Disease 2019 (COVID-19) pandemic. FDA is committed to providing timely guidance to support response efforts to this pandemic.
One investigational treatment being explored for COVID-19 is the use of convalescent plasma collected from individuals who have recovered from COVID-19. Convalescent plasma that contains antibodies to severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2 (the virus that causes COVID-19) is being studied for administration to patients with COVID-19. Use of convalescent plasma has been studied in outbreaks of other respiratory infections, including the 2003 SARS-CoV-1 epidemic, the 2009-2010 H1N1 influenza virus pandemic, and the 2012 MERS-CoV epidemic.
Although promising, convalescent plasma has not yet been shown to be safe and effective as a treatment for COVID-19. Therefore, it is important to study the safety and efficacy of COVID-19 convalescent plasma in clinical trials.
Pathways for Use of Investigational COVID-19 Convalescent Plasma
The following pathways are available for administering or studying the use of COVID-19 convalescent plasma:
Clinical Trials
Investigators wishing to study the use of convalescent plasma in a clinical trial should submit requests to FDA for investigational use under the traditional IND regulatory pathway (21 CFR Part 312). CBER’s Office of Blood Research and Review is committed to engaging with sponsors and reviewing such requests expeditiously. During the COVID-19 pandemic, INDs may be submitted via email to CBERDCC_eMailSub@fda.hhs.gov.
Expanded Access
An IND application for expanded access is an alternative for use of COVID-19 convalescent plasma for patients with serious or immediately life-threatening COVID-19 disease who are not eligible or who are unable to participate in randomized clinical trials (21 CFR 312.305). FDA has worked with multiple federal partners and academia to open an expanded access protocol to facilitate access to COVID-19 convalescent plasma across the nation. Access to this investigational product may be available through participation of acute care facilities in an investigational expanded access protocol under an IND that is already in place.
Although participation in clinical trials or an expanded access program are ways for patients to obtain access to convalescent plasma, for various reasons these may not be readily available to all patients in potential need. Therefore, given the public health emergency that the COVID-19 pandemic presents, and while clinical trials are being conducted and a national expanded access protocol is available, FDA also is facilitating access to COVID-19 convalescent plasma for use in patients with serious or immediately life-threatening COVID-19 infections through the process of the patient’s physician requesting a single patient emergency IND (eIND) for the individual patient under 21 CFR 312.310. This process allows the use of an investigational drug for the treatment of an individual patient by a licensed physician upon FDA authorization, if the applicable regulatory criteria are met. Note, in such case, a licensed physician seeking to administer COVID-19 convalescent plasma to an individual patient must request the eIND (see 21 CFR 312.310(b)).
Today, the U.S. Food and Drug Administration issued an emergency use authorization (EUA) for investigational convalescent plasma for the treatment of COVID-19 in hospitalized patients as part of the agency’s ongoing efforts to fight COVID-19. Based on scientific evidence available, the FDA concluded, as outlined in its decision memorandum, this product may be effective in treating COVID-19 and that the known and potential benefits of the product outweigh the known and potential risks of the product.
Today’s action follows the FDA’s extensive review of the science and data generated over the past several months stemming from efforts to facilitate emergency access to convalescent plasma for patients as clinical trials to definitively demonstrate safety and efficacy remain ongoing.
The EUA authorizes the distribution of COVID-19 convalescent plasma in the U.S. and its administration by health care providers, as appropriate, to treat suspected or laboratory-confirmed COVID-19 in hospitalized patients with COVID-19.
Alex Azar, Health and Human Services Secretary:
“The FDA’s emergency authorization for convalescent plasma is a milestone achievement in President Trump’s efforts to save lives from COVID-19,” said Secretary Azar. “The Trump Administration recognized the potential of convalescent plasma early on. Months ago, the FDA, BARDA, and private partners began work on making this product available across the country while continuing to evaluate data through clinical trials. Our work on convalescent plasma has delivered broader access to the product than is available in any other country and reached more than 70,000 American patients so far. We are deeply grateful to Americans who have already donated and encourage individuals who have recovered from COVID-19 to consider donating convalescent plasma.”
Stephen M. Hahn, M.D., FDA Commissioner:
“I am committed to releasing safe and potentially helpful treatments for COVID-19 as quickly as possible in order to save lives. We’re encouraged by the early promising data that we’ve seen about convalescent plasma. The data from studies conducted this year shows that plasma from patients who’ve recovered from COVID-19 has the potential to help treat those who are suffering from the effects of getting this terrible virus,” said Dr. Hahn. “At the same time, we will continue to work with researchers to continue randomized clinical trials to study the safety and effectiveness of convalescent plasma in treating patients infected with the novel coronavirus.”
Scientific Evidence on Convalescent Plasma
Based on an evaluation of the EUA criteria and the totality of the available scientific evidence, the FDA’s Center for Biologics Evaluation and Research determined that the statutory criteria for issuing an EUA criteria were met.
The FDA determined that it is reasonable to believe that COVID-19 convalescent plasma may be effective in lessening the severity or shortening the length of COVID-19 illness in some hospitalized patients. The agency also determined that the known and potential benefits of the product, when used to treat COVID-19, outweigh the known and potential risks of the product and that that there are no adequate, approved, and available alternative treatments.
CLINICAL MEMORANDUM From: , OBRR/DBCD/CRS To: , OBRR Through: , OBRR/DBCD , OBRR/DBCD , OBRR/DBCD/CRS Re: EUA 26382: Emergency Use Authorization (EUA) Request (original request 8/12/20; amended request 8/23/20) Product: COVID-19 Convalescent Plasma Items reviewed: EUA request Fact Sheet for Health Care Providers Fact Sheet for Recipients Sponsor: Robert Kadlec, M.D. Assistant Secretary for Preparedness and Response (ASPR) Office of Assistant Secretary for Preparedness and Response (ASPR) U.S. Department of Health and Human Services (HHS) EXECUTIVE SUMMARY COVID-19 Convalescent Plasma (CCP), an unapproved biological product, is proposed for use under an Emergency Use Authorization (EUA) under section 564 of the Federal Food, Drug, and Cosmetic Act (the Act),(21 USC 360bbb-3) as a passive immune therapy for the treatment of hospitalized patients with COVID-19, a serious or life-threatening disease. There currently is no adequate, approved, and available alternative to CCP for treating COVID-19. The sponsor has pointed to four lines of evidence to support that CCP may be effective in the treatment of hospitalized patients with COVID-19: 1) History of convalescent plasma for respiratory coronaviruses; 2) Evidence of preclinical safety and efficacy in animal models; 3) Published studies of the safety and efficacy of CCP; and 4) Data on safety and efficacy from the National Expanded Access Treatment Protocol (EAP) sponsored by the Mayo Clinic. Considering the totality of the scientific evidence presented in the EUA, I conclude that current data for the use of CCP in adult hospitalized patients with COVID-19 supports the conclusion that CCP meets the “may be effective” criterion for issuance of an EUA from section 564(c)(2)(A) of the Act. It is reasonable to conclude that the known and potential benefits of CCP outweigh the known and potential risks of CCP for the proposed EUA. Current data suggest the largest clinical benefit is associated with high-titer units of CCP administered early course of the disease.
A letter, from Senator Warren, to Commissioner Hahn from Senate Committee asking for documentation for any communication between FDA and White House
August 25, 2020 Dr. Stephen M. Hahn, M.D. Commissioner of Food and Drugs U.S. Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993 Dear Commissioner Hahn: We write regarding the U.S. Food and Drug Administration’s (FDA) troubling decision earlier this week to issue an Emergency Use Authorization (EUA) for convalescent plasma as a treatment for coronavirus disease 2019 (COVID-19).1 Reports suggests that the FDA granted the EUA amid intense political pressure from President Trump and other Administration officials, despite limited evidence of convalescent plasma’s effectiveness as a COVID-19 treatment.2 To help us better understand whether the issuance of the blood plasma EUA was motivated by politics, we request copies of any and all communications between FDA and White House officials regarding the blood plasma EUA.
The authorization will allow health-care providers in the U.S. to use the plasma to treat hospitalized patients with Covid-19.
The FDA’s emergency use authorization came a day after President Trump accused the agency of delaying enrollment in clinical trials for vaccines or therapeutics.
The criticism from Trump and action from the FDA led some scientists to believe the authorization, which came on the eve of the GOP national convention, was politically motivated.
FDA Commissioner Dr. Stephen Hahn is walking back comments on the benefits of convalescent plasma, saying he could have done a better job of explaining the data on its effectiveness against the coronavirus after authorizing it for emergency use over the weekend.
In an interview with Bloomberg’s Drew Armstrong, FDA Commissioner Hahn reiterates that his decision was based on hard evidence and scientific fact, not political pressure. The whole interview is at the link below:
Dr. Hahn corrected his initial statement about 35% of people would be cured by convalescent plasma. In the interview he stated:
I was trying to do what I do with patients, because patients often understand things in absolute terms versus relative terms. And I should’ve been more careful, there’s no question about it. What I was trying to get to is that if you look at a hundred patients who receive high titre, and a hundred patients who received low titre, the difference between those two particular subset of patients who had these specific criteria was a 35% reduction in mortality. So I frankly did not do a good job of explaining that.
FDA colleagues had frank discussion after the statement was made. He is not asking for other people in HHS to retract their statements, only is concerned that FDA has correct information for physicians and patients
Hahn is worried that people will not enroll due to chance they may be given placebo
He gave no opinion when asked if FDA should be an independent agency
For more articles on COVID19 please go to our Coronavirus Portal at
RNA from the SARS-CoV-2 virus taking over the cells it infects: Virulence – Pathogen’s ability to infect a Resistant Host: The Imbalance between Controlling Virus Replication versus Activation of the Adaptive Immune Response
Curator: Aviva Lev-Ari, PhD, RN – I added colors and bold face
UPDATED on 9/8/2020
What bats can teach us about developing immunity to Covid-19 | Free to read
Another duality and paradox in the Treatment of COVID-19 Patients in ICUs was expressed by Mike Yoffe, MD, PhD, David H. Koch Professor of Biology and Biological Engineering, Massachusetts Institute of Technology. Dr. Yaffe has a joint appointment in Acute Care Surgery, Trauma, and Surgical Critical Care, and in Surgical Oncology @BIDMC
on 6/29 at SOLUTIONS with/in/sight at Koch Institute @MIT
How Are Cancer Researchers Fighting COVID-19? (Part II)”Jun 29, 2020 11:30 AM EST
In COVID-19 patients: two life threatening conditions are seen in ICUs:
Blood Clotting – Hypercoagulability or Thrombophilia
Cytokine Storm – immuno-inflammatory response
The coexistence of 1 and 2 – HINDERS the ability to use effectively tPA as an anti-clotting agent while the cytokine storm is present.
Mike Yoffe’s related domain of expertise:
Signaling pathways and networks that control cytokine responses and inflammation
Misregulation of cytokine feedback loops, along with inappropriate activation of the blood clotting cascade causes dysregulation of cell signaling pathways in innate immune cells (neutrophils and macrophages), resulting in tissue damage and multiple organ failure following trauma or sepsis. Our research is focused on understanding the role of the p38-MK2 pathway in cytokine control and innate immune function, and on cross-talk between cytokines, clotting factors, and neutrophil NADPH oxidase-derived ROS in tissue damage, coagulopathy, and inflammation, using biochemistry, cell biology, and mouse knock-out/knock-in models. We recently discovered a particularly important link between abnormal blood clotting and the complement pathway cytokine C5a which causes excessive production of extracellular ROS and organ damage by neutrophils after traumatic injury.
SARS-CoV-2 infection induces low IFN-I and -III levels with a moderate ISG response
Strong chemokine expression is consistent across in vitro, ex vivo, and in vivo models
Low innate antiviral defenses and high pro-inflammatory cues contribute to COVID-19
Summary
Viral pandemics, such as the one caused by SARS-CoV-2, pose an imminent threat to humanity. Because of its recent emergence, there is a paucity of information regarding viral behavior and host response following SARS-CoV-2 infection. Here we offer an in-depth analysis of the transcriptional response to SARS-CoV-2 compared with other respiratory viruses. Cell and animal models of SARS-CoV-2 infection, in addition to transcriptional and serum profiling of COVID-19 patients, consistently revealed a unique and inappropriate inflammatory response. This response is defined by low levels of type I and III interferons juxtaposed to elevated chemokines and high expression of IL-6. We propose that reduced innate antiviral defenses coupled with exuberant inflammatory cytokine production are the defining and driving features of COVID-19.
Defining the Transcriptional Response to SARS-CoV-2 Relative to Other Respiratory Viruses
To compare the transcriptional response of SARS-CoV-2 with other respiratory viruses, including MERS-CoV, SARS-CoV-1, human parainfluenza virus 3 (HPIV3), respiratory syncytial virus (RSV), and IAV, we first chose to focus on infection in a variety of respiratory cell lines (Figure 1). To this end, we collected poly(A) RNA from infected cells and performed RNA sequencing (RNA-seq) to estimate viral load. These data show that virus infection levels ranged from 0.1% to more than 50% of total RNA reads (Figure 1A).
Discussion
In the present study, we focus on defining the host response to SARS-CoV-2 and other human respiratory viruses in cell lines, primary cell cultures, ferrets, and COVID-19 patients. In general, our data show that the overall transcriptional footprint of SARS-CoV-2 infection was distinct in comparison with other highly pathogenic coronaviruses and common respiratory viruses such as IAV, HPIV3, and RSV. It is noteworthy that, despite a reduced IFN-I and -III response to SARS-CoV-2, we observed a consistent chemokine signature. One exception to this observation is the response to high-MOI infection in A549-ACE2 and Calu-3 cells, where replication was robust and an IFN-I and -III signature could be observed. In both of these examples, cells were infected at a rate to theoretically deliver two functional virions per cell in addition to any defective interfering particles within the virus stock that were not accounted for by plaque assays. Under these conditions, the threshold for PAMP may be achieved prior to the ability of the virus to evade detection through production of a viral antagonist. Alternatively, addition of multiple genomes to a single cell may disrupt the stoichiometry of viral components, which, in turn, may itself generate PAMPs that would not form otherwise. These ideas are supported by the fact that, at a low-MOI infection in A549-ACE2 cells, high levels of replication could also be achieved, but in the absence of IFN-I and -III induction. Taken together, these data suggest that, at low MOIs, the virus is not a strong inducer of the IFN-I and -III system, as opposed to conditions where the MOI is high.
Taken together, the data presented here suggest that the response to SARS-CoV-2 is imbalanced with regard to controlling virus replication versus activation of the adaptive immune response. Given this dynamic, treatments for COVID-19 have less to do with the IFN response and more to do with controlling inflammation. Because our data suggest that numerous chemokines and ILs are elevated in COVID-19 patients, future efforts should focus on U.S. Food and Drug Administration (FDA)-approved drugs that can be rapidly deployed and have immunomodulating properties.
One of the features distinguishing SARS-CoV-2 from its more pathogenic counterpart SARS-CoV is the presence of premature stop codons in its ORF3b gene. Here, we show that SARS-CoV-2 ORF3b is a potent interferon antagonist, suppressing the induction of type I interferon more efficiently than its SARS-CoV ortholog. Phylogenetic analyses and functional assays revealed that SARS-CoV-2-related viruses from bats and pangolins also encode truncated ORF3b gene products with strong anti-interferon activity. Furthermore, analyses of more than 15,000 SARS-CoV-2 sequences identified a natural variant, in which a longer ORF3b reading frame was reconstituted. This variant was isolated from two patients with severe disease and further increased theability of ORF3b to suppress interferon induction. Thus, our findings not only help to explain the poor interferon response in COVID-19 patients, but also describe a possibility of the emergence of natural SARS-CoV-2 quasi-species with extended ORF3b that may exacerbate COVID-19 symptoms.
Highlights
ORF3b of SARS-CoV-2 and related bat and pangolin viruses is a potent IFN antagonist
SARS-CoV-2 ORF3b suppresses IFN induction more efficiently than SARS-CoV ortholog
The anti-IFN activity of ORF3b depends on the length of its C-terminus
An ORF3b with increased IFN antagonism was isolated from two severe COVID-19 cases
RNA (in green) from the SARS-CoV-2 virus is shown taking over the cells it infects.ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
A deep dive into how the new coronavirus infects cells has found that it orchestrates a hostile takeover of their genes unlike any other known viruses do, producing what one leading scientist calls “unique” and “aberrant” changes.Recent studies show that in seizing control of genes in the human cells it invades, the virus changes how segments of DNA are read, doing so in a way that might explain why the elderly are more likely to die of Covid-19 and why antiviral drugs might not only save sick patients’ lives but also prevent severe disease if taken before infection.“It’s something I have never seen in my 20 years of” studying viruses, said virologist Benjamin tenOever of the Icahn School of Medicine at Mount Sinai, referring to how SARS-CoV-2, the virus that causes Covid-19, hijacks cells’ genomes.The “something” he and his colleagues saw is how SARS-CoV-2 blocks one virus-fighting set of genes but allows another set to launch, a pattern never seen with other viruses. Influenza and the original SARS virus (in the early 2000s), for instance, interfere with both arms of the body’s immune response — what tenOever dubs “call to arms” genes and “call for reinforcement” genes.The first group of genes produces interferons. These proteins, which infected cells release, are biological semaphores, signaling to neighboring cells to activate some 500 of their own genes that will slow down the virus’ ability to make millions of copies of itself if it invades them. This lasts seven to 10 days, tenOever said, controlling virus replication and thereby buying time for the second group of genes to act.This second set of genes produce their own secreted proteins, called chemokines, that emit a biochemical “come here!” alarm. When far-flung antibody-making B cells and virus-killing T cells sense the alarm, they race to its source. If all goes well, the first set of genes holds the virus at bay long enough for the lethal professional killers to arrive and start eradicating viruses.
“Most other viruses interfere with some aspect of both the call to arms and the call for reinforcements,” tenOever said. “If they didn’t, no one would ever get a viral illness”: The one-two punch would pummel any incipient infection into submission.
SARS-CoV-2, however, uniquely blocks one cellular defense but activates the other, he and his colleagues reported in a study published last week in Cell. They studied healthy human lung cells growing in lab dishes, ferrets (which the virus infects easily), and lung cells from Covid-19 patients. In all three, they found that within three days of infection, the virus induces cells’ call-for-reinforcement genes to produce cytokines. But it blocks their call-to-arms genes — the interferons that dampen the virus’ replication.
The result is essentially no brakes on the virus’s replication, but a storm of inflammatory molecules in the lungs, which is what tenOever calls an “unique” and “aberrant” consequence of how SARS-CoV-2 manipulates the genome of its target.
In another new study, scientists in Japan last week identified how SARS-CoV-2 accomplishes that genetic manipulation. Its ORF3b gene produces a protein called a transcription factor that has “strong anti-interferon activity,” Kei Sato of the University of Tokyo and colleagues found — stronger than the original SARS virus or influenza viruses. The protein basically blocks the cell from recognizing that a virus is present, in a way that prevents interferon genes from being expressed.
In fact, the Icahn School team found no interferons in the lung cells of Covid-19 patients. Without interferons, tenOever said, “there is nothing to stop the virus from replicating and festering in the lungs forever.”
That causes lung cells to emit even more “call-for-reinforcement” genes, summoning more and more immune cells. Now the lungs have macrophages and neutrophils and other immune cells “everywhere,” tenOever said, causing such runaway inflammation “that you start having inflammation that induces more inflammation.”
At the same time, unchecked viral replication kills lung cells involved in oxygen exchange. “And suddenly you’re in the hospital in severe respiratory distress,” he said.
In elderly people, as well as those with diabetes, heart disease, and other underlying conditions, the call-to-arms part of the immune system is weaker than in younger, healthier people, even before the coronavirus arrives. That reduces even further the cells’ ability to knock down virus replication with interferons, and imbalances the immune system toward the dangerous inflammatory response.
The discovery that SARS-CoV-2 strongly suppresses infected cells’ production of interferons has raised an intriguing possibility: that taking interferons might prevent severe Covid-19 or even prevent it in the first place, said Vineet Menachery of the University of Texas Medical Branch.
In a study of human cells growing in lab dishes, described in a preprint (not peer-reviewed or published in a journal yet), he and his colleagues also found that SARS-CoV-2 “prevents the vast amount” of interferon genes from turning on. But when cells growing in lab dishes received the interferon IFN-1 before exposure to the coronavirus, “the virus has a difficult time replicating.”
After a few days, the amount of virus in infected but interferon-treated cells was 1,000- to 10,000-fold lower than in infected cells not pre-treated with interferon. (The original SARS virus, in contrast, is insensitive to interferon.)
Ending the pandemic and preventing its return is assumed to require an effective vaccine to prevent infectionand antiviral drugs such as remdesivir to treat the very sick, but the genetic studies suggest a third strategy: preventive drugs.
It’s possible that treatment with so-called type-1 interferon “could stop the virus before it could get established,” Menachery said.
Giving drugs to healthy people is always a dicey proposition, since all drugs have side effects — something considered less acceptable than when a drug is used to treat an illness. “Interferon treatment is rife with complications,” Menachery warned. The various interferons, which are prescribed for hepatitis, cancers, and many other diseases, can cause flu-like symptoms.
But the risk-benefit equation might shift, both for individuals and for society, if interferons or antivirals or other medications are shown to reduce the risk of developing serious Covid-19 or even make any infection nearly asymptomatic.
Interferon “would be warning the cells the virus is coming,” Menachery said, so such pretreatment might “allow treated cells to fend off the virus better and limit its spread.” Determining that will of course require clinical trials, which are underway.
Other related articles in this Open Access Online Scientific Journal include the following:
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
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
Glycobiology vs Proteomics: Glycobiologists Prespective in the effort to explain the origin, etiology and potential therapeutics for the Coronavirus Pandemic (COVID-19).
Actemra, immunosuppressive which was designed to treat rheumatoid arthritis but also approved in 2017 to treat cytokine storms in cancer patients SAVED the sickest of all COVID-19 patients
The Accelerating COVID-19 Therapeutic Interventions and Vaccines (ACTIV) Partnership on May 18, 2020: Leadership of AbbVie, Amgen, AstraZeneca, Bristol Myers Squibb, Eisai, Eli Lilly, Evotec, Gilead, GlaxoSmithKline, Johnson & Johnson, KSQ Therapeutics, Merck, Novartis, Pfizer, Roche, Sanofi, Takeda, and Vir. We also thank multiple NIH institutes (especially NIAID), the FDA, BARDA, CDC, the European Medicines Agency, the Department of Defense, the VA, and the Foundation for NIH
Tweets & Retweets 2020 World Medical Innovation Forum – COVID-19, AI and the Future of Medicine, Featuring Harvard and Industry Leader Insights – MGH & BWH, Virtual Event: Monday, May 11, 8:15 a.m. – 5:15 p.m. ET
A Series of Recently Published Papers Report the Development of SARS-CoV2 Neutralizing Antibodies and Passive Immunity toward COVID19
Curator: Stephen J. Williams, Ph.D.
Passive Immunity and Treatment of Infectious Diseases
The ability of one person to pass on immunity to another person (passive immunity) is one of the chief methods we develop immunity to many antigens. For instance, maternal antibodies are passed to the offspring in the neonatal setting as well as in a mother’s milk during breast feeding. In the clinical setting this is achieved by transferring antibodies from one patient who has been exposed to an antigen (like a virus) to the another individual. However, the process of purifying the most efficacious antibody as well as its mass production is limiting due to its complexity and cost and can be prohibitively long delay during a pandemic outbreak, when therapies are few and needed immediately. Regardless, the benefits of developing neutralizing antibodies to confer passive immunity versus development of a vaccine are evident, as the former takes considerable less time than development of a safe and effective vaccine. For a good review on the development and use of neutralizing antibodies and the use of passive immunity to treat infectious diseases please read the following review:
Antibodies have been used for over a century in the prevention and treatment of infectious disease. They are used most commonly for the prevention of measles, hepatitis A, hepatitis B, tetanus, varicella, rabies, and vaccinia. Although their use in the treatment of bacterial infection has largely been supplanted by antibiotics, antibodies remain a critical component of the treatment of diptheria, tetanus, and botulism. High-dose intravenous immunoglobulin can be used to treat certain viral infections in immunocompromised patients (e.g., cytomegalovirus, parvovirus B19, and enterovirus infections). Antibodies may also be of value in toxic shock syndrome, Ebola virus, and refractory staphylococcal infections. Palivizumab, the first monoclonal antibody licensed (in 1998) for an infectious disease, can prevent respiratory syncytial virus infection in high-risk infants. The development and use of additional monoclonal antibodies to key epitopes of microbial pathogens may further define protective humoral responses and lead to new approaches for the prevention and treatment of infectious diseases.
TABLE 1
Summary of the efficacy of antibody in the prevention and treatment of infectious diseases
A Great Explanation of Active versus Passive Immunity by Dr. John Campbell, one of the pioneers in the field of immunology:Antibodies have been used for over a century in the prevention and treatment of infectious disease. They are used most commonly for the prevention of measles, hepatitis A, hepatitis B, tetanus, varicella, rabies, and vaccinia. Although their use in the treatment of bacterial infection has largely been supplanted by antibiotics, antibodies remain a critical component of the treatment of diptheria, tetanus, and botulism. High-dose intravenous immunoglobulin can be used to treat certain viral infections in immunocompromised patients (e.g., cytomegalovirus, parvovirus B19, and enterovirus infections). Antibodies may also be of value in toxic shock syndrome, Ebola virus, and refractory staphylococcal infections. Palivizumab, the first monoclonal antibody licensed (in 1998) for an infectious disease, can prevent respiratory syncytial virus infection in high-risk infants. The development and use of additional monoclonal antibodies to key epitopes of microbial pathogens may further define protective humoral responses and lead to new approaches for the prevention and treatment of infectious diseases.
However, developing successful neutralizing antibodies can still be difficult but with the latest monoclonal antibody technology, as highlighted by the following papers, this process has made much more efficient. In addition, it is not feasable to isolate antibodies from the plasma of covalescent patients in a scale that is needed for a worldwide outbreak.
When fighting off foreign invaders, our bodies make antibodies precisely produced for the task. The reason vaccines offer such long-lasting protection is they train the immune system to identify a pathogen, so immune cells remember and are ready to attack the virus when it appears. Monoclonal antibodies for coronavirus would take the place of the ones our bodies might produce to fight the disease. The manufactured antibodies would be infused into the body to either tamp down an existing infection, or to protect someone who has been exposed to the virus. However, these drugs are synthetic versions of the convalescent plasma treatments that rely on antibodies from people who have recovered from infection. But the engineered versions are easier to scale because they’re manufactured in rats, rather than from plasma donors.
The following papers represent the latest published work on development of therapeutic and prophylactic neutralizing antibodies to the coronavirus SARS-CoV2
1. Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody.
SARS-CoV-2 is a newly emerged coronavirus responsible for the current COVID-19 pandemic that has resulted in more than 3.7 million infections and 260,000 deaths as of 6 May 20201,2. Vaccine and therapeutic discovery efforts are paramount to curb the pandemic spread of this zoonotic virus. The SARS-CoV-2 spike (S) glycoprotein promotes entry into host cells and is the main target of neutralizing antibodies. Here we describe multiple monoclonal antibodies targeting SARS-CoV-2 S identified from memory B cells of an individual who was infected with SARS-CoV in 2003. One antibody, named S309, potently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2 by engaging the S receptor-binding domain. Using cryo-electron microscopy and binding assays, we show that S309 recognizes a glycan-containing epitope that is conserved within the sarbecovirus subgenus, without competing with receptor attachment. Antibody cocktails including S309 along with other antibodies identified here further enhanced SARS-CoV-2 neutralization and may limit the emergence of neutralization-escape mutants. These results pave the way for using S309- and S309-containing antibody cocktails for prophylaxis in individuals at high risk of exposure or as a post-exposure therapy to limit or treat severe disease.
2. Potent neutralizing antibodies against SARS-CoV-2 identified by high-throughput single-cell sequencing of convalescent patients’ B cells
The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here we report the rapid identification of SARS-CoV-2 neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes, 14 potent neutralizing antibodies were identified with the most potent one, BD-368-2, exhibiting an IC50 of 1.2 ng/mL and 15 ng/mL against pseudotyped and authentic SARS-CoV-2, respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally, the 3.8Å Cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody’s epitope overlaps with the ACE2 binding site. Moreover, we demonstrated that SARS-CoV-2 neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV neutralizing antibodies. Altogether, we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B-cell sequencing in response to pandemic infectious diseases.
3. A human monoclonal antibody blocking SARS-CoV-2 infection
The emergence of the novel human coronavirus SARS-CoV-2 in Wuhan, China has caused a worldwide epidemic of respiratory disease (COVID-19). Vaccines and targeted therapeutics for treatment of this disease are currently lacking. Here we report a human monoclonal antibody that neutralizes SARS-CoV-2 (and SARS-CoV) in cell culture. This cross-neutralizing antibody targets a communal epitope on these viruses and may offer potential for prevention and treatment of COVID-19.
Extra References on Development of Neutralizing antibodies for COVID19 {Sars-CoV2} published this year (2020) [1-4]
Fan P, Chi X, Liu G, Zhang G, Chen Z, Liu Y, Fang T, Li J, Banadyga L, He S et al: Potent neutralizing monoclonal antibodies against Ebola virus isolated from vaccinated donors. mAbs 2020, 12(1):1742457.
Dussupt V, Sankhala RS, Gromowski GD, Donofrio G, De La Barrera RA, Larocca RA, Zaky W, Mendez-Rivera L, Choe M, Davidson E et al: Potent Zika and dengue cross-neutralizing antibodies induced by Zika vaccination in a dengue-experienced donor. Nature medicine 2020, 26(2):228-235.
Young CL, Lyons AC, Hsu WW, Vanlandingham DL, Park SL, Bilyeu AN, Ayers VB, Hettenbach SM, Zelenka AM, Cool KR et al: Protection of swine by potent neutralizing anti-Japanese encephalitis virus monoclonal antibodies derived from vaccination. Antiviral research 2020, 174:104675.
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