Tag Archives: #antigen

New Corona Mass Test Up to 100 Times More Sensitive Than Rapid Antigen Tests (Medicine)

A new corona test developed at the University Hospital Bonn can analyze a large number of swabs simultaneously using sequencing technology and has a similarly high sensitivity as the common qPCR test. The innovative method offers great potential, especially for systematic testing in daycare centers, schools or companies. Today, the results of the study on the new Corona test have been published in the renowned journal Nature Biotechnology.

Bonn, 6/29/2021 – In addition to vaccination, systematic testing of the population remains of central importance in order to effectively monitor and contain the spread of infections during the Coronavirus pandemic. Only in this way can the spread of the virus be effectively monitored and contained through targeted measures.

The innovative corona test “LAMP-Seq”, which has been developed at the University Hospital Bonn (UKB), offers the possibility to test many people regularly for the SARS-CoV-2 virus. In this way, infections can be detected at an early stage and corresponding chains of infection can be interrupted quickly. “Our corona test “LAMP-Seq” can detect about 100 times lower amounts of virus than current rapid antigen tests and is almost as sensitive and specific as the common qPCR test” describes Prof. Dr. Jonathan Schmid-Burgk from the Institute of Clinical Chemistry and Clinical Pharmacology of the UKB the characteristics of the test procedure, which was developed interdisciplinary with other researchers at the UKB.

“Added to this is the high scalability of the test. By using sequencing machines, thousands of samples can be analyzed simultaneously,” said Schmid-Burgk, who was appointed to the University of Bonn from the Broad Institute of MIT and Harvard in early 2020. The LAMP-Seq method detects not only corona infections with the original SARS-CoV-2 virus, but also the novel variants of concern alpha to delta.

A microreaction vessel (often referred to as an “eppi”) filled with 1 milliliter of liquid contains the amplified swab material from up to 10,000 corona tests, which are analyzed with a sequencer. © Felix Heyder / University Hospital Bonn

Members of the ImmunoSensation2 cluster of excellence, the Institute of Hygiene and Public Health, Life&Brain GmbH and Bundeswehr Central Hospital Koblenz were among those involved in the project.

For the “LAMP-Seq” test, the Bonn scientists have adapted the already established LAMP method (“Loop-mediated Isothermal Amplification” – propagation of the viral genome at a constant temperature) and made it compatible with sequencing machines used for biomedical research. As a result, many samples can be analyzed simultaneously in a high-throughput procedure. Before thousands of samples are analyzed together in a sequencing run, each individual sample is linked to a molecular barcode. This barcode ensures that each sample can be assigned without doubt, even after thousands of samples have been pooled. “Retesting of the entire pool in case of a positive test result is therefore no longer necessary” says Dr. Kerstin Ludwig, Emmy-Noether group leader at the Institute of Human Genetics. This technology significantly reduces the cost per test in comparison to the qPCR test and makes the “LAMP-Seq” procedure a scalable corona mass test.

“With its high throughput and sensitivity, the “LAMP-Seq” test can make a significant contribution to the screening of undetected infections. Especially in schools or companies, where many people regularly meet, the corona test is ideal to systematically and preventively monitor the occurrence of infections” describes Ludwig, the co-developer of the test procedure, the possible application scenarios of the “LAMP-Seq” test.

Prof. Wolfgang Holzgreve, Medical Director and CEO of the UKB, explains the benefits of the new test for corona surveillance as follows: “In order to effectively contain a pandemic, infected people must be found before they infect others. To achieve this goal, we need mass screenings with the highest sensitivity that can give us a detailed picture of existing chains of infection. This is exactly what the corona test “LAMP-Seq” developed at UKB is suited for.”

Even smaller models of the sequencing machines used are capable of analyzing around 10,000 samples in a single run (duration: around ten to twelve hours). This virtually eliminates laboratory capacity as a limiting factor in testing.

In several large studies (including school and employee testing) with a total of around 20,000 tests, the Bonn scientists have extensively tested, optimized and successfully validated the entire upstream and downstream logistics, from sample collection by throat swabs to fully digital feedback of the test results. The documentation of the study results recently passed an independent peer review process and was published today in the renowned journal Nature Biotechnology.

While the Bonn scientists have currently focused their innovative method entirely on SARS-CoV-2 testing, “LAMP-Seq” can also be used in the future for differential diagnostics in testing for other viruses such as influenza A and can also be quickly adapted to other viruses.

The scientists are currently working on CE certification in order to make the “LAMP-Seq” test available internationally in the near future. Until this approval is obtained, the technically and scientifically fully validated “LAMP-Seq” method will continue to be used for pilot testing.

The scientific team received financial support from the German Federal Ministry of Education and Research BMBF within the framework of the “Bundesweites Forschungsnetz Angewandte Surveillance und Testung” (B-FAST).

Featured image: Blue-stained swab material from corona tests is prepared for analysis in a sequencing device with the help of a laboratory robot. © Felix Heyder / University Hospital Bonn


Publication: Kerstin U. Ludwig, Ricarda M. Schmithausen, David Li, Max L. Jacobs, Ronja Hollstein, Katja Blumenstock, Jana Liebing, Miko?aj S?abicki, Amir Ben-Shmuel, Ofir Israeli, Shay Weiss, Thomas S. Ebert, Nir Paran, Wibke Rüdiger, Gero Wilbring, David Feldman, Bärbel Lippke, Nina Ishorst, Lara M. Hochfeld, Eva C. Beins, Ines H. Kaltheuner, Maximilian Schmitz, Aliona Wöhler, Manuel Döhla, Esther Sib, Marius Jentzsch, Jacob D. Borrajo, Jonathan Strecker, Julia Reinhardt, Brian Cleary, Matthias Geyer, Michael Hölzel, Rhiannon Macrae, Markus M. Nöthen, Per Hoffmann, Martin Exner, Aviv Regev, Feng Zhang, Jonathan L. Schmid-Burgk: “LAMP-Seq enables sensitive, multiplexed COVID-19 diagnostics using molecular barcoding”, Nature Biotechnology 2021, Internet: https://www.nature.com/articles/s41587-021-00966-9


Provided by University Hospital Bonn

A Rapid Antigen Test For SARS-CoV-2 in Saliva (Medicine)

Scientists from Hokkaido University have shown that an antigen-based test for quantifying SARS-CoV-2 in saliva samples is simple, rapid, and more conducive for mass-screening.

More than a year into the COVID-19 pandemic, the RT-PCR test remains the gold standard for detection of the SARS-CoV-2 virus. This method requires trained personnel at every step, from collection of nasopharyngeal swab (NPS) samples to interpretation of the results; in addition, the entire process ranges from 24-48 hours on average. As the virus can be transmitted by an infected person before symptoms develop, and is even transmitted by individuals who are asymptomatic, the ability to screen a large number of people quickly is vital to controlling and preventing the spread of the pandemic. Faster methods to detect the SARS-CoV-2 antigens have been developed, but they are not as sensitive as the RT-PCR test. In June 2020, a novel antigen-based kit, Lumipulse® SARS-CoV-2 Ag kit (Lumipulse), was developed by Fujirebio to quantitatively measure the viral antigen in biological samples within 35 minutes.

A team of scientists from Hokkaido University have used the antigen kit to detect SARS-CoV-2 in saliva samples, and have assessed the efficiency and accuracy of the test compared to RT-PCR. Their findings show that the antigen detection kit, which is used to perform chemiluminescent enzyme immunoassay (CLEIA), can rapidly detect SARS-CoV-2 with good accuracy in these samples. The study was published in the journal The Lancet Microbe.

The scientists tested 2056 individuals from three cohorts: patients with clinically confirmed COVID-19, individuals who had contacted patients with COVID-19, and individuals tested on arrival at Tokyo and Kansai International Airports. Saliva samples were collected from all individuals and used for RT-PCR tests as well as CLEIA using Lumipulse. The results of both were compared to determine the usefulness of CLEIA.

The CLEIA test (y-axis) for SARS-CoV-2 correlates well with the RT-PCR test (x-axis). Orange circles indicate symptomatic cases and blue crosses indicate asymptomatic cases. (Isao Yokota, et al. The Lancet Microbe. May 19, 2021).

The scientists found that CLEIA is a reliable test, as it correlates well with RT-PCR. CLEIA alone can be used to detect SARS-CoV-2 within an hour; however, using CLEIA for screening and RT-PCR for confirmation increases the accuracy of diagnosis. 

The benefit of using saliva samples is the ease of collection: it is quick and can be collected by the individuals being tested, reducing the risk that healthcare workers are exposed to the virus. Furthermore, self-collection of saliva allows multiple samples to be collected simultaneously for expeditious screening of visitors at large gatherings.

Combined CLEIA and RT-PCR testing on saliva samples has already been implemented at Japanese airport quarantines, and the authors recommend adopting it at a wider scale to rapidly screen for SARS-CoV-2.

Peter Y. Shane, Takanori Teshima and Isao Yokota, key contributors to this work (Photo: Naoki Namba).

Original Article:

Isao Yokota, et al. A novel strategy for SARS-CoV-2 mass-screening using quantitative antigen testing of saliva: a diagnostic accuracy studyThe Lancet Microbe. May 19, 2021.

Funding:

This study was supported by a Health, Labour and Welfare Policy Research Grant (20HA2002) from the Ministry of Health, Labour and Welfare, Japan.

Featured image: The Lumipulse G600II instrument (left) and the Lumipulse® SARS-CoV-2 Ag kit (right), both manufactured by Fujirebio, which were used in this study for the quantification of SARS-CoV-2 in saliva samples (Photo: Shinichi Fujisawa).


Provided by Hokkaido University

TalTech Scientists Developed Novel Immune Diagnostics of Multiple Sclerosis (Medicine)

Multiple sclerosis (MS) is the most common neurological disease in young adults, affecting more than 2 million individuals worldwide, with about 1500 cases in Estonia. About 20% of MS patients experience optic neuritis (ON) as the presenting symptom, but not all ON patients develop MS.

The TalTech gene technology research unit, in collaboration with the laboratory of Protobios OÜ and medical researchers of the University of Helsinki, published their findings in the prestigious journal of EBioMedicine entitled “Identification of two highly antigenic epitope markers predicting multiple sclerosis in optic neuritis patients”. The lead author Helle Sadam and co-authors Mariliis Jaago and Annika Rähni are the PhD students of the TalTech Department of Chemistry and Biotechnology, Division of Gene Technology.

The principal investigator for the study, Kaia Palm, associate professor in the Division of Gene Technology TalTech and head of research at Protobios OÜ: “We have developed and patented a very powerful technology called Mimotope Variation Analysis (MVA) for the development of diagnostic tests and delineation of novel drug targets. It is based on the recognition of the diversity of the human B-cell immune response or antibody profile. The immune response mediated by B-lymphocytes plays an important role in the development of both (MS and ON) pathologies, so it is a promising target for detecting early diagnostic biomarkers for named diseases.”

Professor Pentti Tienari from the Department of Neurosciences at the University of Helsinki and co-author of the study said, when speaking about the significance of the work: ” Treatment of MS is most effective, when started early, but there have been only few biomarkers available to identify people at risk after the first episode of optic neuritis.”

Professor Antti Vaheri, co-author and Professor Emeritus of Virology, University of Helsinki, added: “Notably, critical involvement of viruses in neurological diseases has therapeutic implications, especially in the case of herpesviruses against which multiple antiviral agents exist.”

In the published work, more than 500 different clinical samples (incl. blood and cerebrospinal fluid samples) from Finnish, as well as Estonian patients were analyzed by MVA. The results provide a broad, high-resolution view on humoral immunity associated with different cases and report on the prognostic value of viral antibodies as novel blood biomarkers for predicting risk of MS after the first episode of ON.

Featured image: Protobios laboratory in action © Tõnu Tuulas


Reference: Helle Sadam, Arno Pihlak, Mariliis Jaago, Nadežda Pupina, Annika Rähni, Maarja Toots, Antti Vaheri, Janne K. Nieminen, Mika Siuko, Pentti J. Tienari, Kaia Palm, Identification of two highly antigenic epitope markers predicting multiple sclerosis in optic neuritis patients, EBioMedicine, Volume 64, 2021, 103211, ISSN 2352-3964, https://doi.org/10.1016/j.ebiom.2021.103211 (https://www.sciencedirect.com/science/article/pii/S2352396421000049)


Provided by Estonian Research Council

The Mechanics of the Immune System (Biology)

When T-cells of our immune system become active, tiny traction forces at the molecular level play an important role. They have now been studied at TU Wien.

Highly complicated processes constantly take place in our body to keep pathogens in check: The T-cells of our immune system are busy searching for antigens – suspicious molecules that fit exactly into certain receptors of the T-cells like a key into a lock. This activates the T-cell and the defense mechanisms of the immune system are set in motion.

Novel microcopy methods allow scientists to study the mechanical interaction of T-cells and particles © Tu Wein

How this process takes place at the molecular level is not yet well understood. What is now clear, however, is that not only chemistry plays a role in the docking of antigens to the T-cell; micromechanical effects are important too. Submicrometer structures on the cell surface act like microscopic tension springs. Tiny forces that occur as a result are likely to be of great importance for the recognition of antigens. At TU Wien, it has now been possible to observe these forces directly using highly developed microscopy methods.

This was made possible by a cooperation between TU Wien, Humbold Universität Berlin, ETH Zurich and MedUni Vienna. The results have now been published in the scientific journal “Nano Letters”.

Smelling and feeling

As far as physics is concerned, our human sensory organs work in completely different ways. We can smell, i.e. detect substances chemically, and we can touch, i.e. classify objects by the mechanical resistance they present to us. It is similar with T cells: they can recognize the specific structure of certain molecules, but they can also “feel” antigens in a mechanical way.

“T cells have so-called microvilli, which are tiny structures that look like little hairs,” says Prof. Gerhard Schütz, head of the biophysics working group at the Institute of Applied Physics at TU Wien. As the experiments showed, remarkable effects can occur when these microvilli come into contact with an object: The microvilli can encompass the object, similar to a curved finger holding a pencil. They can then even enlarge, so that the finger-like protrusion eventually becomes an elongated cylinder, which is turned over the object.

“Tiny forces occur in the process, on the order of less than a nanonewton,” says Gerhard Schütz. One nanonewton corresponds roughly to the weight force that a water droplet with a diameter of one-twentieth of a millimeter would exert.

Force measurement in the hydrogel

Measuring such tiny forces is a challenge. “We succeed by placing the cell together with tiny test beads in a specially developed gel. The beads carry molecules on their surface to which the T cell reacts,” explains Gerhard Schütz. “If we know the resistance that our gel exerts on the beads and measure exactly how far the beads move in the immediate vicinity of the T-cell, we can calculate the force that acts between the T-cell and the beads.”

These tiny forces and the behavior of the microvilli are likely to be important in recognizing the molecules and thus triggering an immune response. “We know that biomolecules such as proteins show different behavior when they are deformed by mechanical forces or when bonds are simply pulled,” says Gerhard Schütz. “Such mechanisms are also likely to play a role in antigen recognition, and with our measurement methods this can now be studied in detail for the first time.”

Reference: M. Aramesh et al., Functionalized Bead Assay to Measure Three-dimensional Traction Forces during T‑cell Activation, Nano Letters, 2020. https://pubs.acs.org/doi/10.1021/acs.nanolett.0c03964

Provided by Vienna University of Technology

Genetically Engineered T Cells Could Lead to Therapies for Autoimmune Diseases (Medicine)

A new study has found that a novel T cell genetically engineered by University of Arizona Health Sciences researchers is able to target and attack pathogenic T cells that cause Type 1 diabetes, which could lead to new immunotherapy treatments.

Scanning electron micrograph of a human T lymphocyte (also called a T cell) from the immune system of a healthy donor. Credit: NIAID

The immune system fights bacteria, viruses and other pathogens by utilizing several types of T cells, all of which have receptors that are specific to particular antigens. On killer T cells, the receptor works in concert with three signaling modules and a coreceptor to destroy the infected cell. Michael Kuhns, Ph.D., an associate professor in the UArizona College of Medicine—Tucson Department of Immunobiology, copied the evolutionary design to engineer a five-module chimeric antigen receptor, or 5MCAR, T cell.

“The 5MCAR was an attempt to figure out if we could build something by biomimicry, using some of evolution’s natural pieces, and redirect T cells to do what we want them to do. We engineered a 5MCAR that would direct killer T cells to target autoimmune T cells that mediate Type 1 diabetes,” said Dr. Kuhns, who is member of the UArizona Cancer Center, BIO5 Institute and Arizona Center on Aging. “So now, a killer T cell will actually recognize another T cell. We flipped T cell-mediated immunity on its head.”

Dr. Kuhns worked with Thomas Serwold, Ph.D., of the Harvard Medical School-affiliated Joslin Diabetes Center, to test the 5MCAR T cells in a non-obese diabetic mouse model with promising results. The findings recently were published in the Proceedings of the National Academy of Sciences.

“When we saw that the 5MCAR T cells completely eliminated the harmful T cells that invaded the pancreas, we were blown away,” Dr. Serwold said. “It was like they hunted them down. That ability is why we think that 5MCAR T cells have tremendous potential for treating diseases like Type 1 diabetes.”

In 2017, the U.S. Food and Drug Administration approved two chimeric antigen receptor (CAR) T cell therapies for specific types of cancer—one for the treatment of children with acute lymphoblastic leukemia and the other for adults with advanced lymphomas. Those CAR T cells focused solely on the receptor, not the surrounding signaling modules or coreceptor.

Dr. Kuhns believes that by mimicking the form and function of a natural T cell, including its complex five-module structure, researchers will be able to more specifically target antigens with greater sensitivity in the future. This type of personalized immunotherapy is a key initiative of the UArizona Health Sciences, as well as a focus of Dr. Kuhns’ lab.

“I’m generally of the belief that evolution converges on related principles to execute related tasks,” Dr. Kuhns said. “Basic research from labs around the world, including ours, has helped us to understand the complex structure and function of the five-module molecular machines that have evolved to drive T cell responses. We think these results show that a biomimetic approach holds promise for CAR engineering.”

Drs. Kuhns and Serwold recently received a bridge grant from the National Institute of Allergy and Infectious Diseases to continue their research into using 5MCAR T cells to prevent autoimmune disease.

“There are many things we don’t yet know about this technology,” Dr. Kuhns said. “What we know is that it works, and it can be very effective in a mouse model of Type 1 diabetes, so that’s great. Now, we have a lot more work to do.”

References: Shio Kobayashi et al, A biomimetic five-module chimeric antigen receptor (5MCAR) designed to target and eliminate antigen-specific T cells, Proceedings of the National Academy of Sciences (2020). DOI: 10.1073/pnas.2012495117 https://www.pnas.org/content/117/46/28950

Provided by University of Arizona

A Semiconductor Chip Detects Antigen Concentrations at 1 Parts Per Quadrillion Molar Mass (Engineering / Electrical & Electronics)

Overview: Associate Professor Kazuhiro Takahashi and Assistant Professor Yong-Joon Choi of the Department of Electrical and Electronic Information Engineering at Toyohashi University of Technology have developed a chip that can sense antigens at one part per quadrillion molar mass. The chip was created using semiconductor micromachining technology. Antigens derived from diseases and present in blood and saliva were adhered onto the surface of a flexibly deformable nanosheet. The amount of force generated during the interaction between adhered antigens was then converted into nanosheet deformation information in order to successfully detect specific antigens. Created with semiconductor technology machined at the millimeter scale, this sensor chip is expected to contribute to telemedicine by functioning as an IoT biosensor that allows antigen and antibody tests to be carried out from home.

IoT sensor captures and detects trace amounts of antigens on a nano-film surface. ©COPYRIGHT (C) TOYOHASHI UNIVERSITY OF TECHNOLOGY. ALL RIGHTS RESERVED.

Details: The measuring device simply and quickly detects diseases using a minuscule amount of blood, urine, saliva, or other bodily fluid, and will be a vital tool for accurately diagnosing diseases, verifying treatment results, and checking for recurrences and metastasis. Research is being conducted into a biosensor that can measure treatment results and pathological reactions by detecting DNA, RNA and proteins contained in such fluid. This technology has recently attracted interest across the globe, with antigen and antibody detection widely used to detect and determine the presence of novel coronavirus infections. Furthermore, among COVID-19 patients, reports suggest that patients with severe symptoms show differences in multiple protein concentrations contained in the blood when compared to those with mild symptoms. By examining such markers, this technology is expected to be used to predict illness severity. Current detection devices are not digitalized, and require visual confirmation of color changes using a labeling agent. Reading the wide range of markers is time consuming, and has made implementation for IoT devices difficult. The research team is developing a micro sensor chip that checks for diseases using a flexibly deformable nanosheet made using semiconductor micromachining technology. First, an antibody that catches the targeted antigens is fixed onto the nanosheet, and deformations to a thin film caused by electric repulsions among the adhered antigens is measured. To improve sensitivity to the point where the membrane that the antigens adhere to becomes thin and soft, organic nanosheets two-times softer than semiconductor silicon are used. This is expected to improve sensor sensitivity to a magnitude twice that of conventional silicon-based sensors. In addition, development is continuing on signal detection technology that uses a smartphone camera to detect nanosheet deformation.

With this sensor, which is designed for sensitive changes in adhesion of biomolecules, the antibody must be fixed to the nanosheet in advance in order to capture the antigen, and issues related to film degradation can make this process difficult. The research team optimized density for antibodies to adhere to a nano membrane with adjustable thickness, creating a biosensor that detects only antigens with specifically high sensitivity. Moreover, since it is possible to detect deformation to the nano sheet caused by adhered molecules in real time, the technology is expected to allow for quick detection of disease-derived molecules. The biosensor developed in this project was used in an experiment to detect albumin, a protein contained in blood. The experiment successfully detected one femtogram (15 attomoles in molar concentration) of antigen contained in one milliliter. With the minimum detection limit almost equivalent to that of large-scale detection devices that use labeling agents, this device is expected to allow for ultra-sensitive detection on a portable scale.

Future Outlook: Going forward, the research team plans to conduct trials using semiconductor sensors to detect markers for severe symptoms of COVID-19 infection. In addition to blood detection, chemical sensors are being developed to detect odor and chemical substances. We believe we can contribute to an IoT-based society by making new, small-scale sensor devices a reality. Replacing the probe molecule on the surface of our nanosheet, the technology can be used to detect viruses while also detecting a variety of biomarkers. By making these biosensors common in society, we aim to contribute to telemedicine, allowing doctor diagnoses to be performed from home.

This research was conducted with grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) Scientific Research Fund (Basic Research (B)), the Japan Science and Technology Agency (JST) PRESTO Creation of Innovative Nanoelectronics Combining Materials, Devices, and Systems, and a Future Challenge 2050 grant from the New Energy and Industrial Technology Development Organization (NEDO).

Reference: Yong-Joon Choi, Toshiaki Takahashi, Miki Taki, Kazuaki Sawada, and Kazuhiro Takahashi, Label-free attomolar protein detection using a MEMS optical interferometric surface-stress immunosensor with a freestanding PMMA/parylene-C nanosheet, Biosensors and Bioelectronics, DOI: 10.1016/j.bios.2020.112778 https://www.sciencedirect.com/science/article/abs/pii/S095656632030765X?via%3Dihub

Provided by Toyohashi University of Technology

How The Immune System Remembers Viruses (Medicine)

When a virus enters the body, it is picked up by certain cells of the immune system. They transport the virus to the lymph nodes where they present its fragments, known as antigens, to CD8+ T cells responsible control of viral infections. Each of these cells carries a unique T cell receptor on the surface that can recognize certain antigens. However, only very few T cell receptors match a given viral the antigen.

©gettyimages

To bring the infection under control and maximize the defenses against the virus, these few antigen-specific T cells start dividing rapidly and develop into effector T cells. These kill virus-infected host cells and then die off themselves once the infection is cleared. Some of these short-lived effector cells – according to the generally accepted theory – turn into memory T cells, which persist in the organism long term. In case the same pathogen enters the body again, memory T cells are already present and ready to fight the invader more swiftly and effectively than during the first encounter.

Memory cells and their origin

“Prevailing scientific opinion says that activated T cells first become effector cells and only then gradually develop into memory cells,” says Dr. Veit Buchholz, a specialist in microbiology and working group leader at the Institute for Medical Microbiology, Immunology and Hygiene at TUM. “In our view, however, that isn’t the case. It would mean that the more effector cells are formed after contact with the pathogen, the more numerous the memory cells would become.” However, Buchholz and his colleagues observed a different course of events and have now published their results in the journal Nature Immunology.

“We investigated the antiviral immune responses resulting from individual activated T cells in mice and traced the lineage of the ensuing memory cells using single-cell fate mapping,” reports first author Dr. Simon Grassmann. “Based on these experiments, we were able to show that certain ‘T cell families’ descended from individual cells form up to 1000 times more ‘memory’ than others. However, these long-term dominating T cell families only contributed little to the magnitude of the initial immune response, which was dominated by effector cells derived from other shorter-lived T cell families.”

At the level of individual cells, it therefore became evident that development of effector and memory cells segregates at a much earlier stage than previously believed: “Already in the first week after the confrontation with the pathogen, we saw major differences in the transcriptomes of the detected T cell families,” says Lorenz Mihatsch, also a first author of the study. “Normally at this time of the immune response CD8+ T cells are enriched in molecules that help to kill virus infected cells. However, we found no indication of these cytolytic molecules in the long-term dominating T cell families. Instead, they were already geared exclusively towards memory development at this early stage.”

Optimization of vaccines

These results could help to improve vaccine development in the future, says Veit Buchholz: “To generate an optimal immune response through vaccination, the body needs to produce as many memory cells as possible. For that purpose, it is important to have a precise understanding of how individual T cells are programmed.” Buchholz’s study might also prove useful in helping to recognize sooner whether a new vaccine is effective. “To determine the long-term strength of an immune response, it could be helpful to measure the number of memory precursors within a few days of administering a vaccine,” says Buchholz.

References: Grassmann, S., Mihatsch, L., Mir, J. et al. Early emergence of T central memory precursors programs clonal dominance during chronic viral infection. Nat Immunol (2020). https://doi.org/10.1038/s41590-020-00807-y link: http://dx.doi.org/10.1038/s41590-020-00807-y

Provided by Technical University Of Munich

Scientists Set A Trap For Pandemic Causing Viruses (Medicine)

A research team led by Nagoya University scientists in Japan has developed an approach that can quickly find synthetic proteins that specifically bind to important targets, such as components of the SARS-CoV-2 virus. The method was published in the journal Science Advances and could be used to develop test kits or for finding treatments.

The TRAP display method “fishes” for synthetic proteins from among a library of trillions for those that can target SARS-CoV-2. The approach was able to identify proteins that can be used for testing for the virus and potentially treating people infected with COVID-19. ©Hiroshi Murakami

“We developed a laboratory technique for rapid selection of synthetic proteins that strongly bind to SARS-CoV-2,” says Nagoya University biomolecular engineer Hiroshi Murakami. “High-affinity synthetic proteins can be used to develop sensitive antigen tests for SARS-CoV-2 and for future use as neutralization antibodies in infected patients.”

Murakami and his colleagues had previously developed a protein selection lab test called TRAP display, which stands for ‘transcription-translation coupled with association of puromycin linker.’ Their approach skips two time-consuming steps in another commonly used technique for searching through synthetic protein libraries. But their investigations indicated there was a problem with the puromycin linker.

In the current study, the team improved their technique by modifying the puromycin linker. Ultimately, they were able to use their TRAP display to identify nine synthetic proteins that bind to the spike protein on SARS-CoV-2’s outer membrane. The approach took only four days compared to the weeks it would take using the commonly used messenger RNA display technology.

TRAP display involves using a large number of DNA templates that code for and synthesize trillions of proteins carrying random peptide sequences. The synthetic proteins are linked to DNA with the help of the modified puromycin linker and then exposed to a target protein. When the whole sample is washed, only the synthetic proteins that bind to the target remain. These are then placed back into the TRAP display for further rounds until only a small number of very specific target-binding synthetic proteins are left.

The researchers investigated the nine synthetic proteins that were found to bind to SARS-CoV-2. Some were specifically able to detect SARS-CoV-2 in nasal swabs from COVID-19 patients, indicating they could be used in test kits. One also attaches to the virus to prevent it from binding to the receptors it uses to gain access to human cells. This suggests this protein could be used as a treatment strategy.

“Our high-speed, improved TRAP display could be useful for implementing rapid responses to subspecies of SARS-CoV-2 and to other potential new viruses causing future pandemics,” says Murakami.

References: T. Kondo, Y. Iwatani, K. Matsuoka, T. Fujino, S. Umemoto, Y. Yokomaku, K. Ishizaki, S. Kito, T. Sezaki, G. Hayashi, H. Murakami, “Antibody-like proteins that capture and neutralize SARS-CoV-2”, Science Advances 18 Sep 2020: eabd3916 DOI: 10.1126/sciadv.abd3916 link: https://advances.sciencemag.org/content/early/2020/09/18/sciadv.abd3916

Provided by Nagoya University

Researchers Discovered Potential Cause Of Immunotherapy-Related Neurotoxicity (Neuroscience)

New research has uncovered the previously unknown presence of CD19—a B cell molecule targeted by chimeric antigen receptor (CAR) T cell immunotherapy to treat leukemia, lymphoma, and multiple myeloma—in brain cells that protect the blood brain barrier (BBB).

This discovery may potentially be the cause for neurotoxicity in patients undergoing CD19 directed CAR T cell immunotherapy, according to the research team led by Avery Posey, Ph.D., an assistant professor of Systems Pharmacology and Translational Therapeutics in the Perelman School of Medicine at the University of Pennsylvania and Research Health Science Specialist at the Corporal Michael J. Crescenz VA Medical Center in Philadelphia, PA. The study was published today in Cell.

“Our work has revealed that there is CD19 expression in a subset of cells that are, one, not B cells, and two, potentially related to the neurotoxicity we observe in patients treated with CAR T cell therapy targeting CD19,” Posey said. “The next question is, can we identify a better target for eliminating B cell related malignancies other than CD19, or can we engineer around this brain cell expression of CD19 and build a CAR T cell that makes decisions based on the type of cell it encounters—for instance, CAR T cells that kill the B cells they encounter, but spare the CD19 positive brain cells?”

As so often happens in scientific endeavors, the path to this discovery was made somewhat by chance. Kevin Parker, a Ph.D. student at Stanford and co-author on the paper, was at home analyzing previously published single cell sequencing data sets in his spare time. He found CD19 expression in a data set of fetal brain samples that looked odd, because the accepted wisdom was that CD19 only existed in B cells. So his lab reached out to the pioneers of CAR T cell immunotherapy, Penn Medicine.

“I suggested we test this as a preclinical model. When we treated the mouse model with CAR T cells targeting the mouse version of CD19, we found what looks like the start of neurotoxicities,” Posey said.

The team observed an increase in BBB permeability when mouse CD19 was targeted by CAR T cells, even in mice that lack B cells, but not when human CD19 was targeted as a control treatment (mice do not express human CD19).

“Even more interesting, this BBB permeability was more severe when the CAR T cells were fueled by a costimulatory protein called CD28 than when the CAR T cells used 4-1BB,” Posey said “This difference in the severity of BBB permeability correlates with what we know about the clinical observations of CAR T cell-related neurotoxicities—the frequency of patients experiencing high-grade neurotoxicity is lower for those that received the 4-1BB-based CAR T cells.”

His team sought to investigate the higher incidence of neurotoxicity in CD19-directed immunotherapies, compared to treatments targeting other B cell proteins, such as CD20. Notably, CD19 CAR T cells are sensitive to even low levels of CD19 antigen density, emphasizing the importance of identifying any potential reservoir of CD19 other than B cells.

The researchers’ discovery of CD19 molecules in the brain provides evidence that this increase in neurotoxicity is due to CD19-directed CAR T cell immunotherapies. Posey said, though, that generally this neurotoxicity is temporary and patients recover.

This research also highlights the potential utility of developing a comprehensive human single-cell atlas for clinical medicine. Sequencing is an unbiased, genome-wide measurement of gene expression that can capture even rare populations of cells. These rare cell types might otherwise be missed in measurements of bulk tissue due to their low frequency, but as this study demonstrates could be critically important in understanding the clinical effects of targeted therapy. While current CAR T cells recognize only a single antigen, future generations of CAR T cells may be able to discriminate between unique combinations of target antigens to improve thei

r cell-type specificity. The researchers envision that a comprehensive database of gene expression across all human cell types will enable the precise identification of cell type-specific target antigens which can be used to design safe and effective cellular immunotherapies.

“That’s what we think one of the biggest take-home messages is,” Posey said. “The incredible usefulness of single cell atlas or single cell sequencing technology to determine whether a potential immunotherapy or drug target is going to be present somewhere in the body that we would not normally expect it based on conventional thought and whether this expression may lead to toxicity.”

CD19 is thought to be a lineage-restricted molecule—behaving in a functionally and structurally limited way. But this study shows that some small percentage of brain cells also express CD19.

“We would not have identified that through bulk sequencing, where we’re looking at a population of cells versus a single cell type,” Posey said. “It’s only through single cell sequencing that we’re able to identify that there’s this very small percentage of cells in the brain that also contain this molecule, contrary to popular thought.”

References: Kevin R. Parker et al. Single-Cell Analyses Identify Brain Mural Cells Expressing CD19 as Potential Off-Tumor Targets for CAR-T Immunotherapies. Cell. Published: September 21, 2020. DOI:doi.org/10.1016/j.cell.2020.08.022 link: https://www.cell.com/cell/fulltext/S0092-8674(20)31013-8?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867420310138%3Fshowall%3Dtrue