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Microbiology

Memory B cell marker predicts long-lived antibody response to flu vaccine

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Memory B cells play a critical role to provide long-term immunity after a vaccination or infection. In a study published in the journal Immunity, researchers describe a distinct and novel subset of memory B cells that predict long-lived antibody responses to influenza vaccination in humans.

These effector memory B cells appear to be poised for a rapid serum antibody response upon secondary challenge one year later, Anoma Nellore, M.D., Fran Lund, Ph.D., and colleagues at the University of Alabama at Birmingham and Emory University report. Evidence from transcriptional and epigenetic profiling shows that the cells in this subset differ from all previously described memory B cell subsets.

The UAB researchers identified the novel subset by the presence of FcRL5 receptor protein on the cell surface. In immunology, a profusion of different cell-surface markers is used to identify and separate immune-cell types. In the novel memory B cell subset, FcRL5 acts as a surrogate marker for positive expression of the T-bet transcription factor inside the cells. Various transcription factors act as master regulators to orchestrate the expression of many different gene sets as various cell types grow and differentiate.

Nellore, Lund and colleagues found that the FcRL5+ T-bet+ memory B cells can be detected seven days after immunization, and the presence of these cells correlates with vaccine antibody responses months later. Thus, these cells may represent an early, easily monitored cellular compartment that can predict the development of a long-lived antibody response to vaccines.

This could be a boon to the development of a more effective yearly influenza vaccine. “New annual influenza vaccines must be tested, and then manufactured, months in advance of the winter flu season,” Lund said. “This means we must make an educated guess as to which flu strain will be circulating the next winter.”

Why are vaccine candidates made so far in advance? Pharmaceutical companies, Lund says, need to wait many weeks after vaccinating volunteers to learn whether the new vaccine elicits a durable immune response that will last for months. “One potential outcome of the current study is we may have identified a new way to predict influenza vaccine durability that would give us an answer in days, rather than weeks or months,” Lund said. “If so, this type of early ‘biomarker’ could be used to test flu vaccines closer to flu season — and moving that timeline might give us a better shot at predicting the right flu strain for the new annual vaccine.”

Seasonal flu kills 290,000 to 650,000 people each year, according to World Health Organization estimates. The global flu vaccine market was more than $5 billion in 2020.

To understand the Immunity study, it is useful to remember what happens when a vaccinated person subsequently encounters a flu virus.

Following exposure to previously encountered antigens, such as the hemagglutinin on inactivated influenza in flu vaccines, the immune system launches a recall response dominated by pre-existing memory B cells that can either produce new daughter cells or cells that can rapidly proliferate and differentiate into short-lived plasmablasts that produce antibodies to decrease morbidity and mortality. These latter B cells are called “effector” memory B cells.

“The best vaccines induce the formation of long-lived plasma cells and memory B cells,” said Lund, the Charles H. McCauley Professor in the UAB Department of Microbiology and director of the Immunology Institute. “Plasma cells live in your bone marrow and make protective antibodies that can be found in your blood, while memory B cells live for many years in your lymph nodes and in tissues like your lungs.

“Although plasma cells can survive for decades after vaccines like the measles vaccine, other plasma cells wane much more quickly after vaccination, as is seen with COVID-19,” Lund said. “If that happens, memory B cells become very important because these long-lived cells can rapidly respond to infection and can quickly begin making antibody.”

In the study, the UAB researchers looked at B cells isolated from blood of human volunteers who received flu vaccines over a span of three years, as well as B cells from tonsil tissue obtained after tonsillectomies.

They compared naïve B cells, FcRL5+ T-bet+ hemagglutinin-specific memory B cells, FcRL5neg T-betneg hemagglutinin-specific memory B cells and antibody secreting B cells, using standard phenotype profiling and single-cell RNA sequencing. They found that the FcRL5+ T-bet+ hemagglutinin-specific memory B cells were transcriptionally similar to effector-like memory cells, while the FcRL5neg T-betneg hemagglutinin-specific memory B cells exhibited stem-like central memory properties.

Antibody-secreting B cells need to produce a lot of energy to churn out antibody production, and they also must turn on processes that protect the cells from some of the detrimental side effects of that intense metabolism, including controlling the dangerous reactive oxygen species and boosting the unfolded protein response.

The FcRL5+ T-bet+ hemagglutinin-specific memory B cells did not express the plasma cell commitment factor, but did express transcriptional, epigenetic and metabolic functional programs that poised these cells for antibody production. These included upregulated genes for energy-intensive metabolic processes and cellular stress responses.

Accordingly, FcRL5+ T-bet+ hemagglutinin-specific memory B cells at Day 7 post-vaccination expressed intracellular immunoglobulin, a sign of early transition to antibody-secreting cells. Furthermore, human tonsil-derived FcRL5+ T-bet+ memory B differentiated more rapidly into antibody-secreting cells in vitro than did FcRL5neg T-betneg hemagglutinin-specific memory B cells.

Lund and Nellore, an associate professor in the UAB Department of Medicine Division of Infectious Diseases, are co-corresponding authors of the study, “A transcriptionally distinct subset of influenza-specific effector memory B cells predicts long-lived antibody responses to vaccination in humans.”

Co-authors with Lund and Nellore are Esther Zumaquero, R. Glenn King, Betty Mousseau, Fen Zhou and Alexander F. Rosenberg, UAB Department of Microbiology; Christopher D. Scharer, Tian Mi, Jeremy M. Boss, Christopher M. Tipton and Ignacio Sanz, Emory University School of Medicine, Atlanta, Georgia; Christopher F. Fucile, UAB Informatics Institute; John E. Bradley and Troy D. Randall, UAB Department of Medicine, Division of Clinical Immunology and Rheumatology; and Stuti Mutneja and Paul A. Goepfert, UAB Department of Medicine Division of Infectious Diseases.

Funding for the work came from National Institutes of Health grants AI125180, AI109962 and AI142737 and from the UAB Center for Clinical and Translational Science.

  • Anoma Nellore, Esther Zumaquero, Christopher D. Scharer, Christopher F. Fucile, Christopher M. Tipton, R. Glenn King, Tian Mi, Betty Mousseau, John E. Bradley, Fen Zhou, Stuti Mutneja, Paul A. Goepfert, Jeremy M. Boss, Troy D. Randall, Ignacio Sanz, Alexander F. Rosenberg, Frances E. Lund. A transcriptionally distinct subset of influenza-specific effector memory B cells predicts long-lived antibody responses to vaccination in humans. Immunity, 2023; DOI: 10.1016/j.immuni.2023.03.001

    • University of Alabama at Birmingham
    Microbiology

    Fomepizole helps overcome antibiotic-resistant pneumonia in mice, study finds

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    Pneumococcal disease leads to over three million hospitalizations and hundreds of thousands of deaths annually. A study publishing March 16 in the open access journal PLOS Biology by Carlos J. Orihuela at the University of Alabama at Birmingham, Alabama, United States, and colleagues suggests that the FDA-approved drug Fomepizole may reduce disease severity in the lungs of mice with some forms of bacterial pneumonia and enhance the efficacy of the antibiotic erythromycin as well.

    Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. While vaccines to protect against the bacteria are available, these vaccines are not effective against all strains, with some versions being especially problematic as they are multidrug-resistant. Currently, there are very limited treatment options for combating multidrug-resistant S. pneumoniae infections.

    In order to test the effects of novel treatments for antibiotic-resistant S. pneumoniae, the researchers conducted a series of experiments with mice. Fomepizole is an FDA-approved drug normally used as an antidote for the ingestion of toxic alcohols (such as methanol or ethylene glycol), and works by inhibiting the enzyme alcohol dehydrogenase. Researchers inoculated mice with a multidrug-resistant S. pneumoniae and tested the effect of fomepizole in a combinatorial treatment with antibiotics. They quantified the bacterial burden in the organs of infected mice, comparing the experimental group with the control group.

    The researchers found that using Fomepizole blocked normal energy production by S. pneumoniae and enhanced the bacteria’s susceptibility to antibiotics and reduced bacterial burden in the lungs of mice with pneumonia. The combination treatment was effective in preventing the development of invasive disease. Future research is needed however, as this novel drug treatment has not been replicated in clinical studies on humans, who may present with complicating factors such as comorbidities, advanced age, or environmental variables that may play a role in disease outcomes.

    Orihuela adds, “Pharmacological targeting of fermentation pathways is a new way to enhance the susceptibility of some bacteriato antimicrobials. Combination treatment of erythromycin and fomepizole, an alcohol dehydrogenase inhibitor, prevented the in vivo dissemination of antibiotic-resistant Streptococcus pneumoniae.”

  • Hansol Im, Madison L. Pearson, Eriel Martinez, Kyle H. Cichos, Xiuhong Song, Katherine L. Kruckow, Rachel M. Andrews, Elie S. Ghanem, Carlos J. Orihuela. Targeting NAD+ regeneration enhances antibiotic susceptibility of Streptococcus pneumoniae during invasive disease. PLOS Biology, 2023; 21 (3): e3002020 DOI: 10.1371/journal.pbio.3002020

    • PLOS
    Microbiology

    Emergence of extensively drug-resistant Shigella sonnei strain in France

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    Shigellosis, a highly contagious diarrheal disease, is caused by Shigella bacteria circulating in industrializing countries but also in industrialized countries. Scientists from the French National Reference Center for Escherichia coli, Shigella and Salmonella at the Institut Pasteur who have been monitoring Shigella in France for several years have detected the emergence of extensively drug-resistant (XDR) strains of Shigella sonnei. Bacterial genome sequencing and case characteristics (with most cases being reported in male adults) suggest that these strains, which originated in South Asia, mainly spread among men who have sex with men (MSM). This observation needs to be taken into account by clinicians and laboratories when testing for sexually transmitted infections (STIs) in MSM, and systematic antibiograms should be performed if a Shigella strain is isolated to improve treatment for patients infected with XDR strains. The results were published in the journal Nature Communications on January 26, 2023.

    Shigellosis is a highly contagious diarrheal disease that spreads through fecal-oral transmission. Among the different types of Shigella, Shigella sonnei is the species that mainly circulates in industrialized countries. Shigella sonnei infections can cause short-term diarrhea (3-4 days) that resolves on its own. Antibiotic treatment is, however, necessary for moderate to severe cases (bloody diarrhea, risk of complications) or to prevent person-to-person transmission in epidemic situations. The acquisition of antibiotic resistance mechanisms by Shigella bacteria therefore restricts therapeutic options.

    In this study, scientists from the National Reference Center for Escherichia coli, Shigella and Salmonella (CNR-ESS) at the Institut Pasteur demonstrate an increase in antibiotic resistance in S. sonnei isolates collected in France over the past 17 years. The study is based on an analysis of more than 7,000 S. sonnei isolates and epidemiological information gathered in connection with national shigellosis surveillance conducted by the CNR-ESS between 2005 and 2021. The CNR-ESS analyzes all the bacterial isolates sent by its network of private and public partner laboratories throughout France. Over this period, isolates described as “extensively drug resistant” (XDR) were identified for the first time in 2015. The scientists then observed that the proportion of XDR isolates, which are resistant to virtually all the antibiotics recommended for treating shigellosis, increased significantly and reached a peak in 2021, when 22.3% of all S. sonnei isolates (99 cases) were XDR.

    Genome sequencing revealed that all these French XDR strains belonged to the same evolutionary lineage, which became resistant to a key antibiotic (ciprofloxacin) in around 2007 in South Asia. In several geographical regions of the world, including France, the strains then acquired different plasmids coding for resistance to other first-line antibiotics (especially third-generation cephalosporins and azithromycin). For severe cases, the only antibiotics that are still effective are carbapenems or colistin, which must be administered intravenously, resulting in more aggressive treatment that requires more complex monitoring in a hospital environment.

    XDR isolates were observed in France in various contexts: in travelers returning from South Asia or South-East Asia, during an outbreak at a school in 2017 (more than 90 cases, leading to school closure; the index case had returned from South-East Asia) and in men who have sex with men (MSM). The latter were infected by an epidemic clone that has been spreading throughout Europe since 2020 but has also been found in North America and Australia. This subgroup of XDR strains circulating in MSM was the most widespread, accounting for 97% of XDR strains in France in 2021.

    Frequent use of antibiotics in South and South-East Asia, together with repeat treatment for STIs in some people potentially exposed to this risk, increase the likelihood of selection of XDR Shigella strains. Further research is needed to understand the different clinical forms of infection, and especially whether there are asymptomatic forms that might cause the bacteria to spread more widely. Therapeutic trials are also crucial to identify effective oral antibiotics for treating these XDR Shigella strains.

  • Sophie Lefèvre, Elisabeth Njamkepo, Sarah Feldman, Corinne Ruckly, Isabelle Carle, Monique Lejay-Collin, Laëtitia Fabre, Iman Yassine, Lise Frézal, Maria Pardos de la Gandara, Arnaud Fontanet, François-Xavier Weill. Rapid emergence of extensively drug-resistant Shigella sonnei in France. Nature Communications, 2023; 14 (1) DOI: 10.1038/s41467-023-36222-8

    • Institut Pasteur
    Microbiology

    Resistant bacteria are a global problem. Now researchers may have found the solution

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    Staphylococcus aureus. You may have had it in connection with a wound infection. In most cases, it will pass without treatment, while severe cases may require antibiotics, which kills the bacteria. This is the case for the majority of the population. In fact, many of us — though we feel perfectly fine — carry staphylococci in the nose, a good, moist environment in which the bacteria thrive.

    However, more and more staphylococci are becoming resistant to antibiotics (also known as multi resistant staphylococcus aureus or MRSA), and these infections can be difficult to treat.

    “Antibiotics resistance is an increasing problem, especially on a global scale. And when you have this relatively simple infection which suddenly cannot be treated with antibiotics, the situation can turn serious, sometimes life-threatening,” says Professor Niels Ødum from the LEO Foundation Skin Immunology Research Center at the University of Copenhagen.

    Therefore, all over the world, a lot of resources are being invested in fighting antibiotics resistance in staphylococcus aureus infections, and a new study among skin lymphoma patients has produced positive results. A new substance called endolysins has proven capable of killing both resistant and non-resistant staphylococcus aureus — without the need for antibiotics. But we will get back to that.

    The discovery is good news to patients with a weak immune system to whom a staphylococcus aureus infection can be serious and, at worst, fatal. But it also adds to the knowledge we have of other forms of treatment.

    “To people who are severely ill with e.g. skin lymphoma, staphylococci can be a huge, sometimes insoluble problem, as many are infected with a type of staphylococcus aureus that is resistant to antibiotics,” says Niels Ødum and adds:

    “That is why we are careful not to give antibiotics to everyone, because we do not want to have to deal with more resistant bacteria. Therefore, it is important that we find new ways of treating — and not the least to prevent — these infections.”

    New substance may be the answer

    In some patients, a staphylococcus aureus will cause the cancer to worsen. And even though antibiotics appear to work in some cases, it is not without its problems.

    “We can tell that giving high doses of antibiotics to patients with serious infections causes their health, skin and cancer symptoms to improve. But once we stop giving them antibiotics, the symptoms and staphylococci quickly return. Patients experience many adverse effects, and some risk getting resistant bacteria,” says Niels Ødum.

    Therefore, treating staphylococcus aureus can be tricky. At worst, cancer patients may die of an infection which doctors are unable to treat.

    And this is where endolysins enter the scene, as this new substance may be part of the solution to antibiotics resistance like MRSA.

    “This particular endolysin is a brand new, artificially produced enzyme that has been improved several times and designed as a new drug,” explains Postdoc Emil Pallesen, who is first author of the study. He adds:

    “The great thing about this enzyme is that it has been designed to penetrate the wall of staphylococcus aureus. This enables it to target and kill the harmful staphylococcus and leave harmless skin bacteria unharmed.”

    And that is what made the researchers decide to test the new substance; they expected it to be able to kill both resistant and non-resistant staphylococcus bacteria.

    “We have been testing the substance on skin samples from patients, and it does appear to kill staphylococcus aureus from patients. Endolysins do not care whether the bacterium is resistant to antibiotics or not, because it does not work in the same way as antibiotics,” says Niels Ødum and adds:

    “The really good news is that our lab tests have showed that endolysins do not just eradicate staphylococcus aureus; they also inhibit their ability to promote cancer growth.”

  • Emil M.H. Pallesen, Maria Gluud, Chella K. Vadivel, Terkild B. Buus, Bob de Rooij, Ziao Zeng, Sana Ahmad, Andreas Willerslev-Olsen, Christian Röhrig, Maria R. Kamstrup, Lene Bay, Lise Lindahl, Thorbjørn Krejsgaard, Carsten Geisler, Charlotte M. Bonefeld, Lars Iversen, Anders Woetmann, Sergei B. Koralov, Thomas Bjarnsholt, Johan Frieling, Mathias Schmelcher, Niels Ødum. Endolysin inhibits skin colonization by patient-derived Staphylococcus aureus and malignant T cell activation in cutaneous T cell lymphoma. Journal of Investigative Dermatology, 2023; DOI: 10.1016/j.jid.2023.01.039

    • University of Copenhagen – The Faculty of Health and Medical Sciences
    Microbiology

    Innovative approach opens the door to COVID nanobody therapies

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    COVID is not yet under control. Despite a bevy of vaccines, monoclonal antibodies, and antivirals, the virus continues to mutate and elude us. One solution that scientists have been exploring since the early days of the pandemic may come in the form of tiny antibodies derived from llamas, which target various parts of the SARS-CoV-2 spike protein.

    In a new study in the Journal of Biological Chemistry, researchers describe a less expensive way to isolate and identify these so-called nanobodies. The findings will make it easier for scientists around the world to try their hand at discovering nanobodies that target SARS-CoV-2 or other viruses. “Our method is more straightforward and less expensive than existing techniques,” says Rockefeller’s Michael P. Rout. “You do need a llama, but that — along with all the most complicated parts of the process — can be outsourced.”

    The authors have already used this optimized method to identify multiple nanobodies that appear to work against key variants of the virus, including omicron. “COVID is clearly going to be a problem for some time,” Rout says. “We show that many of the nanobodies we have identified with this method target variants-of-concern, so they have real therapeutic potential.”

    Nanobody Novelty

    Nanobodies may work where larger antibodies fail, in part due to their compact size. Studies have shown that nanobodies can squeeze into parts of the SARS-CoV-2 virus that larger antibodies cannot reach. Nanobodies also have unusually long shelf-lives, cost very little to mass-produce and, because of their unique physical properties, could theoretically be inhaled.

    Camelids such as llamas naturally produce nanobodies when exposed to a virus, and Rout and colleagues have developed enormous libraries of promising SARS-CoV-2 nanobodies by giving a small dose of COVID protein to llamas (which produce nanobodies in response, much as humans produce antibodies in response to a vaccine). After taking small blood samples from the llamas and sequencing the nanobody DNA, the scientists later transfer key genes to bacteria which, in turn, produce many more nanobodies for lab analysis.

    But screening these nanobody libraries to see how well they work (and which variants they work against) can be time-consuming and expensive. Rout and colleagues have long relied on the “mass spectrometry” technique, which works extraordinarily well but requires substantial expertise to perform and expensive equipment. They wondered whether a recently discovered “yeast display method,” which was potentially far less expensive and simpler, could also effectively sort through their nanobody library.

    Rout, in collaboration with Rockefeller’s Fred Cross, started by first optimizing the yeast display method. (The two heads-of-lab took the unusual step of performing most of the benchwork themselves). They then used their optimized method to screen a library of nanobodies that they had previously screened with the mass spectrometry technique. They found that their version of the yeast display method not only identified many of the same nanobody candidates as the other approach, but also identified numerous other candidates that they had missed.

    “The method is not ours,” Cross clarifies. “But we made it simpler.”

    Toward Nanobody Therapy

    The relatively simple and low-cost procedure described in the paper could empower laboratories in low-resource areas to generate nanobodies against SARS-CoV-2, as well as other viruses. “A researcher anywhere in the world, with fairly limited resources, could use this technique,” Rout says. “The llama-related stuff could be FedEx-ed from North America.”

    For COVID, the long-term goal is that techniques such as these will lower the bar for entry into nanobody research and ultimately produce therapies that prevent infection. “How we’d make the therapeutic is unestablished, as yet,” Cross says. “The specificity is there and the activity is there, but we don’t have a drug yet. It’d be nice if we did. Hopefully someday.”

    Because with COVID now transitioning to an endemic disease, novel methods for preventing the infection cannot come soon enough. “New variants become prevalent by evading the immune system,” Cross says. “It’s important to have a fast way to find new nanobodies targeting the variants.”

  • Frederick R. Cross, Peter C. Fridy, Natalia E. Ketaren, Fred D. Mast, Song Li, J. Paul Olivier, Kresti Pecani, Brian T. Chait, John D. Aitchison, Michael P. Rout. Expanding and improving nanobody repertoires using a yeast display method: Targeting SARS-CoV-2. Journal of Biological Chemistry, 2023; 299 (3): 102954 DOI: 10.1016/j.jbc.2023.102954

    • Rockefeller University
    Microbiology

    Looking for risky viruses now to get ahead of future pandemics

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    Most of what scientists know about viruses in animals is the list of nucleotides that compose their genomic sequence — which, while valuable, offers very few hints about a virus’s ability to infect humans.

    Rather than let the next outbreak take the world by surprise, two virologists say in a SciencePerspective article published today (March 10, 2023) that the scientific community should invest in a four-part research framework to proactively identify animal viruses that might infect humans.

    “A lot of financial investment has gone into sequencing viruses in nature and thinking that from sequence alone we’ll be able to predict the next pandemic virus. And I think that’s just a fallacy,” said Cody Warren, assistant professor of veterinary biosciences at The Ohio State University and co-lead author of the article.

    “Experimental studies of animal viruses are going to be invaluable,” he said. “By measuring properties in them that are consistent with human infection, we can better identify those viruses that pose the greatest risk for zoonosis and then study them further. I think that’s a realistic way of looking at things that should also be considered.”

    Warren co-authored the opinion piece with Sara Sawyer, professor of molecular, cellular and developmental biology at the University of Colorado Boulder.

    One key message Warren and Sawyer want to get across is that knowing an animal virus can attach to a human cell receptor doesn’t paint the whole picture of its zoonotic potential.

    They propose a series of experiments to assess an animal virus’s potential to infect a human: If it is found to enter human cells, can it use those host cells to make copies of itself and multiply? After viral particles are produced, can they get past human innate immunity? And have human immune systems ever been exposed to another virus from the same family?

    Answering these questions could enable scientists to put a pre-zoonotic candidate virus “on the shelf” for further research — perhaps developing a quick way to diagnose the virus in humans if an unattributable illness surfaces and testing existing antivirals as possible treatments, Warren said.

    “Where it becomes difficult is that there may be many animal viruses out there with signatures of human compatibility,” he said. “So which ones do you pick and choose to prioritize for further study? That’s something that needs to be carefully considered.”

    A decent starting point, he and Sawyer suggest, would be operating on the assumption that viruses with the most risk to humans come from “repeat offender” viral families currently infecting mammals and birds. Those include coronaviruses, orthomyxoviruses (influenza) and filoviruses (causing hemorrhagic diseases like Ebola and Marburg). In 2018, the Bombali virus — a new ebolavirus — was detected in bats in Sierra Leone, but its potential to infect humans remains unknown.

    And then there are arteriviruses, such as the simian hemorrhagic fever virus that exists in wild African monkeys, which Sawyer and Warren recently determined has decent potential to spill over to humans because it can replicate in human cells and subvert immune cells’ ability to fight back.

    The 2020 worldwide lockdown to prevent the spread of COVID-19 is still a fresh and painful memory, but Warren notes that the terrible outcomes of the emergence of SARS-CoV-2 could have been much worse. The availability of vaccines within a year of that lockdown was possible only because scientists had spent decades studying coronaviruses and knew how to attack them.

    “So if we invest in studying animal viruses early and understand their biology in more detail, then in the case that they were to emerge in humans later, we’d be better poised to combat them,” Warren said.

    “We are continually going to be exposed to the viruses of animals. Things are never going to change if we stay on the same trajectory,” he said. “And if we stay complacent and only study those animal viruses after they jump into humans, we’re constantly going to be working backwards. We’ll always be behind.”

  • Cody J. Warren, Sara L. Sawyer. Identifying animal viruses in humans: Experimental virology can inform strategic monitoring for new viruses in humans. Science, 2023; 379 (6636): 982-983 DOI: 10.1126/science.ade6985

    • Ohio State University
    Microbiology

    Researchers develop new technology to easily detect active TB

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    A team of faculty from Wayne State University has discovered new technology that will quickly and easily detect active Mycobacterium tuberculosis (TB) infection antibodies. Their work, “Discovery of Novel Transketolase Epitopes and the Development of IgG-Based Tuberculosis Serodiagnostics,” was published in a recent edition of Microbiology Spectrum, a journal published by the American Society for Microbiology. The team is led by Lobelia Samavati, M.D., professor in the Center for Molecular Medicine and Genetics in the School of Medicine. Samavati was joined by Jaya Talreja, Ph.D, and Changya Peng, research scientists in Wayne State’s Department of Internal Medicine.

    TB remains a global health threat, with 10 million new cases and 1.7 million deaths annually. According to the latest World Health Organization report, TB is the 13th leading cause of death and the second leading infectious killer after COVID-19. Latent tuberculous infection (LTBI) is considered a reservoir for TB bacteria and subjects can progress to active TB. One-third of the world’s population is infected with TB and, on average, 5 to 10% of those infected with LTBI will develop active TB disease over the course of their lives, usually within the first five years after initial infection.

    The gold standard tests to determine whether an infection is active TB are the sputum smear and culture tests. However, these methods require collecting sputum, which is time consuming, expensive, requires trained personnel and lacks sensitivity. The current conventional tests differentiating LTBI from uninfected controls — such as tuberculin skin tests (TST) and/or interferongamma release assay (IGRA) — do not differentiate between active TB infection and latent TB. Despite advances in rapid molecular techniques for TB diagnostics, there is an unmet need for a simple inexpensive point-of-care (POC), rapid non-sputum-based test.

    Samavati’s research group has worked for more than 15 years to develop technology for detection of antibodies in various respiratory diseases. Her lab has developed a novel non-sputum based technology and has discovered several novel immune-epitopes that differentialy bind to specific immunoglobulin (IgG) in TB-infected subjects. The levels of epitope-specific IgG in seum can differentiate active TB from LTBI subjects, healthy contols and other respiratory diseases. This technology can be used as a simple serum assay non-sputum based serological POC- TB test, which is highly specific and sensitveto diffentiate active TB from LTBI.

    “Previously, we developed a T7 phage antigen display platform and after immunoscreening of large sets of serum samples, identified 10 clones that differentially bind to serum antibody (IgG) of active TB patients differentiating TB from other respiratory diseases,” said Samavati. “One of these high-performance clones had homology to the Transketolase (TKT) enzyme of TB bacteria that is an essential enzyme required for the intracellular growth of the bacteria in a host. We hypothesized that abundance of IgG in sera against the identified novel neoantigen that we named as TKTµ may differentiate between active TB, LTBI and other non-TB granulomatous lung diseases such as sarcoidosis. We developed a novel direct Peptide ELISA test to quantify the levels of IgG in serum samples against TKTµ. We designed two additional overlapping M.tb TKT-peptide homologs with potential antigenicity corresponding to M.tb-specifictransketolase (M.tb-TKT1 and M.tb-TKT3) and hence standardized three Peptide ELISA (TKTμ, M.tb TKT1 and M.tb TKT3) for the TB serodiagnosis.”

    After development and standardization of a direct peptide ELISA for three peptides, the research team tested 292 subjects, and their TKT-peptide ELISA results show that TB patients have significantly higher levels of TKT-specific antibodies compared to patients who were healthy controls and with LTBI. The increased levels of TKT-specific antibodies is presumably associated with growing M.tb bacteria in active TB patients. TKT plays a key role in the switch from the dormancy to proliferative phase and TKT specific IgG may uncover the differences between active TB and LTBI. Thus, IgG-based serodiagnosis of TB with TKT-peptide ELISA is promising.

    Currently, commercially available serological TB tests show poor sensitivity and specificity. The ELISA results obtained with the Wayne State team’s discovered TKT peptides yielded high specificity and sensitivity. Their results show that IgG antibodies against transketolase can discriminate active tuberculosis. 

    “Our TKT peptide ELISA test requires chemically synthesized TKT peptides to coat the wells in the ELISA plate, less than 100µl blood serum sample from patient, detection reagents and an ELISA plate reader,” said Samavati. “We are extremely enthusiastic about our technology and the fact that with a simple test we can differentiate active TB from LTBI and other respiratory diseases. We believe that our method and TKT peptide ELISA can fit the requirements of the World Health Organization and the Centers for Disease Control and Prevention as a POC screening method.”

    The research team has applied a patent application on its technology and is actively seeking companies interested in investing.

    This research was supported by the National Heart, Lung and Blood Institute of the National Institutes of Health, grant numbers 113508 and 148089. The Foundation for Innovative New Diagnostics (FIND, Geneva, Switzerland) provided TB and LTBI samples.

  • Jaya Talreja, Changya Peng, Tuan-Minh Nguyen, Sorin Draghici, Lobelia Samavati. Discovery of Novel Transketolase Epitopes and the Development of IgG-Based Tuberculosis Serodiagnostics. Microbiology Spectrum, 2023; 11 (1) DOI: 10.1128/spectrum.03377-22

    • Wayne State University – Office of the Vice President for Research
    Microbiology

    A quick new way to screen virus proteins for antibiotic properties

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    As conventional antibiotics continue to lose effectiveness against evolving pathogens, scientists are keen to employ the bacteria-killing techniques perfected by bacteriophages, the viruses that infect bacteria.

    One major challenge standing in their way is the difficulty of studying individual bacteriophage (phage) proteins and determining precisely how the virus wields these tools to kill their host bacteria. New research from Lawrence Berkeley National Laboratory (Berkeley Lab) could help speed things along.

    “We developed a high-throughput genetic screening approach that can identify the part of the bacterial cell targeted by a potent type of phage weapon called ‘single-gene lysis proteins,'” said Vivek Mutalik, a staff scientist in Berkeley Lab’s Biosciences Area and co-author on a new study describing the work in Nature Chemical Biology. “With rising antibiotic resistance, we urgently need antibiotic alternatives. Some of the smallest phages that we know of code for single-gene lysis proteins (Sgls), also known as ‘protein antibiotics,’ to inhibit key components of bacterial cell wall production that, when disrupted, consistently kill the cell.”

    There appears to be at least one type of phage for every known strain of bacteria, and they are thought to be the most abundant biological entities on Earth. In fact, there are an estimated 1031 phage particles on the planet right now, or the equivalent of one trillion phages for every grain of sand. Each of these phages evolve alongside their chosen host strain, allowing them to counter bacterial resistance traits, as they arise, with improved biological weaponry.

    This massive abundance, specificity, and efficacy means that there are plenty around to study, and that we should theoretically be able to use phages to control any harmful microbe. Phages are also harmless to non-bacterial cells, another reason they are so appealing as medicines and biocontrol tools.

    The problem arises when trying to isolate a single phage from the environment and determine which microbe it targets and how. Scientists are often unable to assess phage-bacteria battles based on genomic sequence alone or study them in action because many bacteria can’t be cultured in a lab — and even if they could, there’s an inherent catch-22 of needing to know ahead of time which bacteria to culture in order to study the phages that infect and kill them.

    To sidestep these obstacles and identify the cellular targets of Sgls, Mutalik and his colleagues used a technology the team previously invented called Dual-Barcoded Shotgun Expression Library Sequencing (Dub-seq). Dub-seq allows scientists to employ a coded library of DNA fragments to investigate how unknown genes function, and can be applied to complicated environmental samples that contain the DNA of many organisms — no culturing needed. In this study, the authors used six Sgls from six phages that infect different bacteria and identified the part of the bacterial cell wall or supporting molecules that each Sgl attacks. In collaboration with scientists from Texas A&M University, they conducted a detailed characterization of the function of one Sgl.

    This work showed that the Sgl proteins target pathways for cell wall building that arose very early in the evolutionary history of bacteria and are still used by nearly all bacteria (including pathogenic bacteria). Since the Sgl proteins attack such fundamental and ubiquitous targets, they can kill bacteria other than the phage’s target strain — confirming they have great potential as antibiotics.

    “Phages are extraordinary innovators when it comes to destroying bacteria. We’re really excited to uncover novel bacterial pathogen-targeting mechanisms that could be leveraged into therapies,” said first author Benjamin Adler, a postdoctoral fellow in Jennifer Doudna’s lab at UC Berkeley.

    Now that the team has evaluated the Dub-seq approach for tackling this question, they can apply it to the thousands of single-gene lysis producing phages awaiting characterization in environmental samples that the team has collected from the ocean, soils, and even the human gut. The inspiration for the next breakthrough medicine could be in there, waiting.

  • Benjamin A. Adler, Karthik Chamakura, Heloise Carion, Jonathan Krog, Adam M. Deutschbauer, Ry Young, Vivek K. Mutalik, Adam P. Arkin. Multicopy suppressor screens reveal convergent evolution of single-gene lysis proteins. Nature Chemical Biology, 2023; DOI: 10.1038/s41589-023-01269-7

    • DOE/Lawrence Berkeley National Laboratory
    Microbiology

    Pattern recognition system that monitors disease-causing bacteria in C. elegans

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    A study published in Immunity by physician-scientist Read Pukkila-Worley, MD, and MD/PhD students Nicholas D. Peterson and Samantha Y. Tse describes a new manner of detecting microbial infection that intercepts pathogen-derived signals of growth to assess the relative threat of virulent bacteria. A nuclear hormone receptor in the nematode C. elegans senses a toxic metabolite produced by the bacterial pathogen Pseudomonas aeruginosa to activate innate immunity. These data reveal an ancient strategy that informs the origins of pathogen detection and may be among the most primordial forms of immune sensing in animals.

    “Our research adds to our understanding of how hosts differentiate between beneficial and harmful bacteria, which teaches us something important about how our immune systems evolved,” said Dr. Pukkila-Worley, associate professor of medicine.

    Distinguishing potentially harmful pathogens from benign microorganisms is one of the primary functions of the innate immune system in all animals. This is particularly important for nematodes, such as C. elegans — the transparent microscopic worm often used as a model organism to study genetics and gene function — that consume bacteria as their food source.

    Working with Pseudomonas aeruginosa, a bacteria that commonly infects immune-compromised patients in the hospital and is increasingly resistant to standard antibiotic treatments, Pukkila-Worley and colleagues performed a series of genetic screens with mutant bacteria, one-by-one, to see if any impacted the innate immune system response in C. elegans.

    They found that bacteria that cannot produce a specific phenazine metabolite were able to avoid detection by the innate immune system, suggesting that the bacterial phenazine metabolite was sensed to activate innate immunity.

    “This result was intriguing because P. aeruginosa use phenazines for growth and virulence. Thus, the innate immune system can intercept signals produced by bacteria in order to identify bacteria that have grown to dangerous levels and are poised to cause disease,” said Pukkila-Worley.

    Researchers in the Pukkila-Worley lab designed a second experiment to identify the sensor in the host that detects these phenazine metabolites. They discovered that a specialized type of transcription factor, a nuclear hormone receptor, binds the phenazine metabolite and directly activates anti-pathogen defenses.

    “One of the striking things about our results is that C. elegans senses this bacterial metabolite to detect an individual bacterial pathogen in a remarkably specific manner from among its bacterial food,”said Peterson, an MD/PhD student in the Pukkila-Worley lab.

    In humans, pattern-recognition systems in the intestine involving Toll-like receptors scan the physical structure of different bacteria to sense the presence of infectious microorganisms. Nematodes lost pattern-recognition receptors in evolution. Pukkila-Worley and colleagues show that nematodes use nuclear hormone receptors to detect specific pathogen-derived metabolites to activate innate immunity, which represents a new type of pattern-recognition.

    Since C. elegans have 274 nuclear hormone receptors, it’s possible that the nematode genome contains dozens of these metabolite recognition systems. Nuclear hormone receptors are also found in most animals, including humans, suggesting that similar metabolite detection systems might exist in other organisms.

    “It’s remarkable that C. elegans evolved mechanisms to differentiate good and bad bacteria even without canonical receptors for pathogen detection. This further supports the importance of understanding how our immune system evolved over time to deepen our understanding of host-microbiome interactions,” said Tse, an MD/PhD student in the Pukkila-Worley lab.

  • Nicholas D. Peterson, Samantha Y. Tse, Qiuyu Judy Huang, Khursheed A. Wani, Celia A. Schiffer, Read Pukkila-Worley. Non-canonical pattern recognition of a pathogen-derived metabolite by a nuclear hormone receptor identifies virulent bacteria in C. elegans. Immunity, 2023; DOI: 10.1016/j.immuni.2023.01.027

    • UMass Chan Medical School
    Microbiology

    Researchers identify three intestinal bacteria found in dementia with Lewy bodies

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    Dementia with Lewy bodies (DLB), one of the most common forms of dementia, has no cure. Previous studies suggested that gut bacteria, the microorganisms that live in the human digestive tract, play a role in Parkinson’s disease, another neurodegenerative disorder, but the bacteria involved in DLB had not been identified. Now, a group led by researchers at the Nagoya University Graduate School of Medicine in Japan has identified three bacteria involved in DLB: Collinsella, Ruminococcus, and Bifidobacterium. Their findings, reported in npj Parkinson’s Disease, suggest new avenues for diagnosis and treatment.

    The onset of DLB is associated with abnormal deposits of alpha-synuclein, a protein in the brain that plays a role in the transmission of signals between neurons. The presence of these deposits, known as ‘Lewy bodies’, affects chemicals in the brain, leading to declines in thinking, reasoning and memory. Symptoms include confusion, memory loss, impaired movement, and visual hallucinations.

    Parkinson’s disease also starts with movement problems, but some patients develop cognitive decline within one year. These patients are diagnosed with DLB when this cognitive decline occurs. Physicians find it difficult to predict which people with Parkinson’s disease will develop cognitive decline within a year and become patients with DLB.

    A research group led by Associate Professor Masaaki Hirayama (Omics Medicine), Professor Kinji Ohno (Neurogenetics), and Assistant Professor Hiroshi Nishiwaki (Neurogenetics) of Nagoya University Graduate School of Medicine, in collaboration with Okayama Neurology Clinic, Iwate Medical University, and Fukuoka University, analyzed microorganisms in the gut and fecal bile acids of patients with DLB, Parkinson’s disease, and rapid eye movement behavior disorder. They discovered that three intestinal bacteria, Collinsella, Ruminococcus, and Bifidobacterium, were associated with patients with DLB. This may suggest possible ways of diagnosing and treating this neurodegenerative disease.

    The researchers also found similarities between the gut bacteria involved in Parkinson’s disease and DLB. In both diseases, the bacteria Akkermansia, which degrades the intestinal mucosa, increased. On the other hand, the bacteria that produce short-chain fatty acids (SCFA) in the gut decreased. “Decreases in SCFA-producing bacteria have been repeatedly reported in Parkinson’s disease, Alzheimer’s disease, and ALS,” explains Ohno. “This suggests that it is a common feature of neurodegenerative diseases.” SCFA are important because they produce regulatory T cells. These types of cells play a critical role in regulating the immune system by suppressing neuroinflammation.

    On the other hand, in patients with DLB, the researchers found an increase in Ruminococcus torques, an increase in Collinsella, and a decrease in Bifidobacterium. This was different from Parkinson’s disease patients, whose levels did not change. In the future using these insights, doctors may be able to analyze the bacteria in a person’s digestive tract to distinguish DLB from Parkinson’s disease.

    Importantly, the reduced levels of Bifidobacterium mayalsosuggest possible ways to treat DLB. Bifidobacterium increases brain-derived neurotrophic factor, a key protein that supports the growth, development and maintenance of neurons in the central and peripheral nervous systems. Therefore, its decrease in DLB is likely to be associated with cognitive decline.

    Similarly, both Ruminococcus torques and Collinsella are intestinal bacteria that carry an enzyme, the product of which regulates inflammation in a region of the brain called the substantia nigra.The substantia nigra produces dopamine, a neurotransmitter that is involved in the regulation of movement and is deficient in Parkinson’s disease. Compared to Parkinson’s disease, the levels of these bacteria were higher in people with DLB. This may explain why the effect on movement is delayed, a key feature that distinguishes DLB from Parkinson’s disease.

    “Our findings can be used both for both diagnosis and treatment,” explains Ohno. “If a patient with Parkinson’s disease develops dementia in one year after the onset of motor symptoms, they are diagnosed with DLB. However, we cannot currently predict whether a patient with Parkinson’s disease will become a DLB patient. The gut microbiome will help to identify such patients.”

    “In terms of treatment, the administration of Ruminococcus torques and Collinsella in patients with Parkinson’s is expected to delay neuroinflammation in the substantia nigra,” Ohno added. “Therapeutic intervention to increase Bifidobacterium may delay the onset and progression of DLB and reduce cognitive dysfunction.”

    “The presence of intestinal bacteria unique to DLB may explain why some patients develop Parkinson’s disease and others develop DLB first,” Ohno said. “Normalizing the abnormal bacteria shared between DLB and Parkinson’s disease may delay the development of both diseases. Improving the gut microbiota is a stepping stone in the treatment of dementia. Our findings may pave the way for the discovery of new and completely different therapeutics.”

  • Hiroshi Nishiwaki, Jun Ueyama, Kenichi Kashihara, Mikako Ito, Tomonari Hamaguchi, Tetsuya Maeda, Yoshio Tsuboi, Masahisa Katsuno, Masaaki Hirayama, Kinji Ohno. Gut microbiota in dementia with Lewy bodies. npj Parkinson’s Disease, 2022; 8 (1) DOI: 10.1038/s41531-022-00428-2

    • Nagoya University