Tag Archives: Blood

Microbiology

Novel subset of memory B cells predicts long-lived antibody responses to influenza vaccination

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Reviewed by Emily Henderson, B.Sc.Mar 22 2023

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.

Source:

University of Alabama at Birmingham

Journal reference:

Nellore, A., et al. (2023). A transcriptionally distinct subset of influenza-specific effector memory B cells predicts long-lived antibody responses to vaccination in humans. Immunity. doi.org/10.1016/j.immuni.2023.03.001.

Microbiology

Leaving lymph nodes intact until after immunotherapy could boost efficacy against solid tumors

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Reviewed by Emily Henderson, B.Sc.Mar 20 2023

Cancer treatment routinely involves taking out lymph nodes near the tumor in case they contain metastatic cancer cells. But new findings from a clinical trial by researchers at UC San Francisco and Gladstone Institutes shows that immunotherapy can activate tumor-fighting T cells in nearby lymph nodes.

The study, published March 16, 2023 in Cell, suggests that leaving lymph nodes intact until after immunotherapy could boost efficacy against solid tumors, only a small fraction of which currently respond to these newer types of treatments.

Most immunotherapies are aimed only at reinvigorating T cells in the tumor, where they often become exhausted battling the tumor’s cancer cells. But the new research shows that allowing the treatment to activate the immune response of the lymph nodes as well can play an important role in driving positive response to immunotherapy.

This work really changes our thinking about the importance of keeping lymph nodes in the body during treatment.”

Matt Spitzer, PhD, investigator for the Parker Institute for Cancer Immunotherapy and Gladstone-UCSF Institute of Genomic Immunology and senior author of the study

Lymph nodes are often removed because they are typically the first place metastatic cancer cells appear, and without surgery, it can be difficult to determine whether the nodes contain metastases.

“Immunotherapy is designed to jump start the immune response, but when we take out nearby lymph nodes before treatment, we’re essentially removing the key locations where T cells live and can be activated,” Spitzer said, noting that the evidence supporting the removal of lymph nodes is from older studies that predate the use of today’s immunotherapies.

Aim for the lymph nodes, not the tumor

Researchers have largely been working under the assumption that cancer immunotherapy works by stimulating the immune cells within the tumor, Spitzer said. But in a 2017 study in mice, Spitzer showed that immunotherapy drugs are actually activating the lymph nodes.

“That study changed our understanding of how these therapies might be working,” said Spitzer. Rather than the immunotherapy pumping up the T cells in the tumor, he said, T cells in the lymph nodes are likely the source for T cells circulating in the blood. Such circulating cells can then go into the tumor and kill off the cancer cells.

Having shown that intact lymph nodes can temper cancer’s hold in mice, Spitzer’s team wanted to know whether the same would prove true in human patients. They chose to design a trial for patients with head and neck cancers because of the high number of lymph nodes in those areas.

The trial enrolled 12 patients whose tumors hadn’t yet metastasized past the lymph nodes. Typically, such patients would undergo surgery to remove the tumor, followed by other treatments if recommended.

Instead, patients received a single cycle of an immunotherapy drug called atezolizumab (anti-PD-L1) that is produced by Genentech, a sponsor of the trial. A week or two later, Spitzer’s team measured how much the treatment activated the patients’ immune systems.

The treatment also included surgically removing each patient’s tumor and nearby lymph nodes after immunotherapy and analyzing how the immunotherapy affected them.

The team found that, after immunotherapy, the cancer-killing T cells in the lymph nodes began springing into action. They also found higher numbers of related immune cells in the patients’ blood.

Spitzer attributes some of the trial’s success to its design, which allowed the team to get a lot of information from a small number of patients by looking at the tissue before and after surgery and running detailed analyses.

“Being able to collect the tissue from surgery shortly after the patients had been given the drug was a really unique opportunity,” he said. “We were able to see, at the cellular level, what the drug was doing to the immune response.”

That kind of insight would be challenging to get from a more traditional trial in patients with later-stage disease, who would not typically benefit from undergoing surgery after immunotherapy.

Metastases inhibit immune response

Another benefit of the study design was that it allowed researchers to compare how the treatment affected lymph nodes with and without metastases, or a second cancer growth.

“No one had looked at metastatic lymph nodes in this way before,” said Spitzer. “We could see that the metastases impaired the immune response relative to what we saw in the healthy lymph nodes.”

It could be that the T cells in these metastatic nodes were less activated by the therapy, Spitzer said. If so, that could explain, in part, the poor performance of some immunotherapy treatments.

Still, the therapy prompted enough T-cell activity in the metastatic lymph nodes to consider leaving them in for a short period of time until treatment ends. “Removing lymph nodes with metastatic cancer cells is probably still important but taking them out before immunotherapy treatment may be throwing the baby out with the bathwater,” said Spitzer.

A subsequent goal of the current trial is to determine whether giving immunotherapy before surgery protects against the recurrence of tumors in the future. Researchers won’t know the answer to that until they’ve had a chance to monitor the participants for several years.

“My hope is that if we can activate a good immune response before the tumor is taken out, all those T cells will stay in the body and recognize cancer cells if they come back,” Spitzer said.

Next, the team plans to study better treatments for patients with metastatic lymph nodes, using drugs that would be more effective at reactivating their immune responses.

Source:

University of California – San Francisco

Journal reference:

Rahim, M. K., et al. (2023). Dynamic CD8+ T cell responses to cancer immunotherapy in human regional lymph nodes are disrupted in metastatic lymph nodes. Cell. doi.org/10.1016/j.cell.2023.02.021

Microbiology

Avanced genome editing technology could be used as a one-time treatment for CD3 delta SCID

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Reviewed by Emily Henderson, B.Sc.Mar 21 2023

A new UCLA-led study suggests that advanced genome editing technology could be used as a one-time treatment for the rare and deadly genetic disease CD3 delta severe combined immunodeficiency.

The condition, also known as CD3 delta SCID, is caused by a mutation in the CD3D gene, which prevents the production of the CD3 delta protein that is needed for the normal development of T cells from blood stem cells.

Without T cells, babies born with CD3 delta SCID are unable to fight off infections and, if untreated, often die within the first two years of life. Currently, bone marrow transplant is the only available treatment, but the procedure carries significant risks.

In a study published in Cell, the researchers showed that a new genome editing technique called base editing can correct the mutation that causes CD3 delta SCID in blood stem cells and restore their ability to produce T cells.

The potential therapy is the result of a collaboration between the laboratories of Dr. Donald Kohn and Dr. Gay Crooks, both members of the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA and senior authors of the study.

Kohn’s lab has previously developed successful gene therapies for several immune system deficiencies, including other forms of SCID. He and his colleagues turned their attention to CD3 delta SCID at the request of Dr. Nicola Wright, a pediatric hematologist and immunologist at the Alberta Children’s Hospital Research Institute in Canada, who reached out in search of a better treatment option for her patients.

CD3 delta SCID is prevalent in the Mennonite community that migrates between Canada and Mexico.

Because newborns are not screened for SCID in Mexico, I often see babies who have been diagnosed late and are returning to Canada quite sick.”

Dr. Nicola Wright, pediatric hematologist and immunologist at the Alberta Children’s Hospital Research Institute

When Kohn presented Wright’s request to his lab, Grace McAuley, then a research associate who joined the lab at the end of her senior year at UCLA, stepped up with a daring idea.

“Grace proposed we try base editing, a very new technology my lab had never attempted before,” said Kohn, a distinguished professor of microbiology, immunology and molecular genetics, and of pediatrics.

Base editing is an ultraprecise form of genome editing that enables scientists to correct single-letter mutations in DNA. DNA is made up of four chemical bases that are referred to as A, T, C and G; those bases pair together to form the “rungs” in DNA’s double-helix ladder structure.

While other gene editing platforms, like CRISPR-Cas9, cut both strands of the chromosome to make changes to DNA, base editing chemically changes one DNA base letter into another -; an A to a G, for example -; leaving the chromosome intact.

“I had a very steep learning curve in the beginning, when base editing just wasn’t working,” said McAuley, who is now pursuing an M.D.-Ph.D. at UC San Diego and is the study’s co-first author. “But I kept pushing forward. My goal was help get this therapy to the clinic as fast as was safely possible.”

McAuley reached out to the Broad Institute’s David Liu, the inventor of base editing, for advice on how to evaluate the technique’s safety for this particular use. Eventually, McAuley identified a base editor that was highly efficient at correcting the disease-causing genetic mutation.

Because the disease is extremely rare, obtaining patient stem cells for the UCLA study was a significant challenge. The project got a boost when Wright provided the researchers with blood stem cells donated by a CD3 delta SCID patient who was undergoing a bone marrow transplant.

The base editor corrected an average of almost 71% of the patient’s stem cells across three laboratory experiments.

Next, McAuley worked with Dr. Gloria Yiu, a UCLA clinical instructor in rheumatology, to test whether the corrected cells could give rise to T cells. Yiu used artificial thymic organoids, which are stem cell-derived tissue models developed by Crooks’ lab that mimic the environment of the human thymus -; the organ where blood stem cells become T cells.

When the corrected blood stem cells were introduced into the artificial thymic organoids, they produced fully functional and mature T cells.

“Because the artificial thymic organoid supports the development of mature T cells so efficiently, it was the ideal system to show that base editing of patients’ stem cells could fix the defect seen in this disease,” said Yiu, who is also a co-first author of the study.

As a final step, McAuley studied the longevity of the corrected stem cells by transplanting them into a mouse. The corrected cells remained four months after transplant, indicating that base editing had corrected the mutation in true, self-renewing blood stem cells. The findings suggest that corrected blood stem cells could persist long-term and produce the T cells patients would need to live healthy lives.

“This project was a beautiful picture of team science, with clinical need and scientific expertise aligned,” said Crooks, a professor of pathology and laboratory medicine. “Every team member played a vital role in making this work successful.”

The research team is now working with Wright on how to bring the new approach to a clinical trial for infants with CD3 delta SCID from Canada, Mexico and the U.S.

This research was funded by the Jeffrey Modell Foundation, the National Institutes of Health, the Bill and Melinda Gates Foundation, the Howard Hughes Medical Institute, the V Foundation and the A.P. Giannini Foundation.

The therapeutic approach described in this article has been used in preclinical tests only and has not been tested in humans or approved by the Food and Drug Administration as safe and effective for use in humans. The technique is covered by a patent application filed by the UCLA Technology Development Group on behalf of the Regents of the University of California, with Kohn and McAuley listed as co-inventors.

Source:

University of California – Los Angeles Health Sciences

Journal reference:

McAuley, G.E., et al. (2023) Human T cell generation is restored in CD3δ severe combined immunodeficiency through adenine base editing. Cell. doi.org/10.1016/j.cell.2023.02.027.

Microbiology

SARS-CoV-2 infection damages the CD8+ T cell response to vaccination

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Reviewed by Emily Henderson, B.Sc.Mar 20 2023

The magnitude and quality of a key immune cell’s response to vaccination with two doses of the Pfizer-BioNTech COVID-19 vaccine were considerably lower in people with prior SARS-CoV-2 infection compared to people without prior infection, a study has found. In addition, the level of this key immune cell that targets the SARS-CoV-2 spike protein was substantially lower in unvaccinated people with COVID-19 than in vaccinated people who had never been infected. Importantly, people who recover from SARS-CoV-2 infection and then get vaccinated are more protected than people who are unvaccinated. These findings, which suggest that the virus damages an important immune-cell response, were published today in the journal Immunity.

The study was co-funded by the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health, and led by Mark M. Davis, Ph.D. Dr. Davis is the director of the Stanford Institute for Immunity, Transplantation and Infection and a professor of microbiology and immunology at Stanford University School of Medicine in Palo Alto, California. He is also a Howard Hughes Medical Institute Investigator.

Dr. Davis and colleagues designed a very sensitive tool to analyze how immune cells called CD4+ T cells and CD8+ T cells respond to SARS-CoV-2 infection and vaccination. These cells coordinate the immune system’s response to the virus and kill other cells that have been infected, helping prevent COVID-19. The tool was designed to identify T cells that target any of dozens of specific regions on the virus’s spike protein as well as some other viral regions. The Pfizer-BioNTech vaccine uses parts of the SARS-CoV-2 spike protein to elicit an immune response without causing infection.

The investigators studied CD4+ and CD8+ T-cell responses in blood samples from three groups of volunteers. One group had never been infected with SARS-CoV-2 and received two doses of the Pfizer-BioNTech COVID-19 vaccine. The second group had previously been infected with SARS-CoV-2 and received two doses of the vaccine. The third group had COVID-19 and was unvaccinated.

The researchers found that vaccination of people who had never been infected with SARS-CoV-2 induced robust CD4+ and CD8+ T-cell responses to the virus’ spike protein. In addition, these T cells produced multiple types of cell-signaling molecules called cytokines, which recruit other immune cells—including antibody-producing B cells—to fight pathogens. However, people who had been infected with SARS-CoV-2 prior to vaccination produced spike-specific CD8+ T cells at considerably lower levels—and with less functionality—than vaccinated people who had never been infected. Moreover, the researchers observed substantially lower levels of spike-specific CD8+ T cells in unvaccinated people with COVID-19 than in vaccinated people who had never been infected.

Taken together, the investigators write, these findings suggest that SARS-CoV-2 infection damages the CD8+ T cell response, an effect akin to that observed in earlier studies showing long-term damage to the immune system after infection with viruses such as hepatitis C or HIV. The new findings highlight the need to develop vaccination strategies to specifically boost antiviral CD8+ T cell responses in people previously infected with SARS-CoV-2, the researchers conclude.  

Source:

National Institutes of Health

Journal reference:

Gao, F., et al. (2023). Robust T cell responses to Pfizer/BioNTech vaccine compared to infection and evidence of attenuated peripheral CD8+ T cell responses due to COVID-19. Immunity. doi.org/10.1016/j.immuni.2023.03.005.

Microbiology

Usefulness of dried blood spot samples for monitoring HCV infection in people who inject drugs

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Reviewed by Emily Henderson, B.Sc.Mar 17 2023

A study with people who inject drugs evaluated a minimally invasive test based on dried blood spots (DBS) for the monitoring of hepatitis C virus (HCV) infection. The use of DBS samples for HCV RNA detection and genotyping was shown to effectively assess cure after treatment and to differentiate between reinfection and treatment failure. The results support the viability of decentralizing treatment and post-treatment monitoring for people who inject drugs, who frequently face challenges accessing the healthcare system. The study, which has been published in the Journal of Medical Virology, was carried out as part of a project with support from the “Conquering Hepatitis Via Microelimination” (CHIME) programme and a PFIS grant. Investigators from various research institutions collaborated in the project, including the Clinical Virology and New Diagnostic Tools research group, led by Dr Elisa Martró, at Germans Trias i Pujol Research Institute (IGTP) and Dr Sabela Lens from Hospital Clínic’s Viral Hepatitis Group.

Towards elimination of hepatitis

In line with the strategy proposed by the World Health Organization for the elimination of viral hepatitis as a public health threat by 2030, and the Plan for Prevention and Control of Hepatitis in Catalonia, which Dr Martró actively participates in, her group has been focused for years on simplifying the diagnosis of hepatitis C by developing and validating an assay which can detect the virus RNA using DBS samples. These minimally invasive samples can be collected at harm reduction centres or drug dependence care and follow-up centers (known as CAS in Catalan), improving access to hepatitis C diagnosis for vulnerable populations, such as people who inject drugs. While this new test has demonstrated good clinical performance as a diagnostic tool for detecting HCV RNA before treatment in previous studies by the Clinical Virology and New Diagnostic Tools research group, the use of DBS samples had not been evaluated as a test for cure or for detecting reinfection after treatment.

A multidisciplinary research group has been able to pursue a project with a new model of care for hepatitis C, based on point-of-care diagnosis, treatment, and reinfection follow-up at the REDAN La Mina harm reduction centre. Since 2019, approximately 750 individuals who inject drugs have been tested though this initiative, which was designed by Dr Sabela Lens from Hospital Clínic’s Viral Hepatitis Unit, in collaboration with the Clinical Virology and New Diagnostic Tools Research Group at Germans Trias i Pujol Research Institute (IGTP), led by Dr Martró from the Microbiology Service (LCMN) of the Germans Trias i Pujol Hospital (HUGTiP), as well as CEEISCAT and the Public Health Agency of Catalonia. The project had the support of the “Conquering Hepatitis Via Microelimination” (CHIME) programme from Gilead Sciences awarded to Dr Lens, as well as a PFIS grant of the Instituto de Salud Carlos III and the Fondo Social Europeo awarded to Anna Not, who is a member of Dr Martró’s group, and aligns with the World Health Organization’s global health strategy, which aims to eliminate hepatitis C as a public health problem by 2030.

A model of decentralized care

In this project, Dr Martró’s group aimed to evaluate the clinical performance of a previously developed HCV-RNA assay based on DBS, for the assessment of cure and the detection of recurrent viremia after on-site treatment at the harm reduction center, compared to the commercially available HCV-RNA point-of-care test. Furthermore, they sought to assess the possibility of distinguishing between reinfection and treatment failure through HCV genotyping from baseline and follow-up DBS samples. Typically, these assessments (cure and reinfection) are performed using venipuncture blood samples collected at healthcare centres, which can be difficult for people who inject drugs and have often limited access to the healthcare system. The recently published results demonstrate how the collection of DBS samples before and after treatment can simplify these assessments in decentralized test-and-treat programmes.

“The success of the CHIME project lies in the decentralized diagnosis and treatment provided at REDAN La Mina. A nurse trained in hepatology assessments was included in the study to enrol and visit participants. The hepatologists at Hospital Clínic also reviewed each case and prescribed decentralized treatment. Additionally, Dr Martró’s group carried out HCV detection and sequencing from DBS samples collected before and after treatment. This pilot program involves HCV diagnosis on-site in less than an hour, treatment at the same center, and follow-up to assess reinfection”, states Dr Lens.

Detection made easier

Reinfection is common in people who inject drugs and must be treated to prevent further transmission of the virus. During early reinfection, low levels of the virus may be present, making its detection in DBS samples challenging, as they only contain a small amount of blood. Of the 193 DBS samples tested after treatment, the DBS-based assay showed 100% specificity and sensitivity ranging from 84% to 96% based on different relevant viral load cut-offs, and similar rates as a test of cure (three months after treatment). It must be born in mind that among the patients with recurrent viremia after treatment, one tenth had low viral loads. Moreover, HCV genotyping allowed researchers to classify 73% of viremic cases as either reinfection or treatment failure.

Collection of DBS samples was done before antiviral treatment and after treatment if recurrent viremia was detected by the commercially available point-of-care assay. Anna Not, the first author of the article (which will be part of her PhD), explains that it “the use of DBS allowed us to sequence the virus before and after treatment and compare the sequences to determine if the virus was the same (indicating a treatment failure) or if it was different (indicating reinfection). This information enabled the hepatologist to decide on the most appropriate antiviral combination for the second treatment”.

The research shows the potential of using DBS samples for determining cure and differentiating between reinfection and relapse after antiviral treatment for hepatitis C in people who inject drugs. The use of DBS samples makes it possible to decentralize treatment and follow-up, improving access to care for these people. Even so, Dr Martró points out that “a small number of patients had low viral loads, which can hinder the detection of viremia and genotyping in DBS. As a result, repeat testing (e.g. every six months) is advised for individuals who are at risk of HCV reinfection”.

Source:

Germans Trias i Pujol Research Institute

Journal reference:

Not, A., et al. (2023) Usefulness of dried blood spot samples for monitoring hepatitis C treatment outcome and reinfection among people who inject drugs in a test-and-treat program. Journal of Medical Virology. doi.org/10.1002/jmv.28544.

Microbiology

Host immune system forms small lesions in the intestines in response to bacterial infection

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Reviewed by Emily Henderson, B.Sc.Mar 14 2023

Yersinia bacteria cause a variety of human and animal diseases, the most notorious being the plague, caused by Yersinia pestis. A relative, Yersinia pseudotuberculosis, causes gastrointestinal illness and is less deadly but naturally infects both mice and humans, making it a useful model for studying its interactions with the immune system.

These two pathogens, as well as a third close cousin, Y. enterocolitica, which affects swine and can cause food-borne illness if people consume infected meat, have many traits in common, particularly their knack for interfering with the immune system’s ability to respond to infection.

The plague pathogen is blood-borne and transmitted by infected fleas. Infection with the other two depends on ingestion. Yet the focus of much of the work in the field had been on interactions of Yersinia with lymphoid tissues, rather than the intestine. A new study of Y. pseudotuberculosis led by a team from Penn’s School of Veterinary Medicine and published in Nature Microbiology demonstrates that, in response to infection, the host immune system forms small, walled-off lesions in the intestines called granulomas. It’s the first time these organized collections of immune cells have been found in the intestines in response to Yersinia infections.

The team went on to show that monocytes, a type of immune cell, sustain these granulomas. Without them, the granulomas deteriorated, allowing the mice to be overtaken by Yersinia.

“Our data reveal a previously unappreciated site where Yersinia can colonize and the immune system is engaged,” says Igor Brodsky, senior author on the work and a professor and chair of pathobiology at Penn Vet. “These granulomas form in order to control the bacterial infection in the intestines. And we show that if they don’t form or fail to be maintained, the bacteria are able to overcome the control of the immune system and cause greater systemic infection.”

The findings have implications for developing new therapies that leverage the host immune system, Brodsky says. A drug that harnessed the power of immune cells to not only keep Yersinia in check but to overcome its defenses, they say, could potentially eliminate the pathogen altogether.

A novel battlefield

Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica share a keen ability to evade immune detection.

“In all three Yersinia infections, a hallmark is that they colonize lymphoid tissues and are able to escape immune control and replicate, cause disease, and spread,” Brodsky says.

Earlier studies had shown that Yersinia prompted the formation of granulomas in the lymph nodes and spleen but had never observed them in the intestines until Daniel Sorobetea, a research fellow in Brodsky’s group, took a closer look at the intestines of mice infected with Y. pseudotuberculosis.

“Because it’s an orally acquired pathogen, we were interested in how the bacteria behaved in the intestines,” Brodsky says. “Daniel made this initial observation that, following Yersinia pseudotuberculosis infection, there were macroscopically visible lesions all along the length of the gut that had never been described before.”

The research team, including Sorobetea and later Rina Matsuda, a doctoral student in the lab, saw that these same lesions were present when mice were infected with Y. enterocolitica, forming within five days after an infection.

A biopsy of the intestinal tissues confirmed that the lesions were a type of granuloma, known as a pyogranuloma, composed of a variety of immune cells, including monocytes and neutrophils, another type of white blood cell that is part of the body’s front line in fighting bacteria and viruses.

Granulomas form in other diseases that involve chronic infection, including tuberculosis, for which Y. pseudotuberculosis is named. Somewhat paradoxically, these granulomas-;while key in controlling infection by walling off the infectious agent-;also sustain a population of the pathogen within those walls.

The team wanted to understand how these granulomas were both formed and maintained, working with mice lacking monocytes as well as animals treated with an antibody that depletes monocytes. In the animals lacking monocytes “these granulomas, with their distinct architecture, wouldn’t form,” Brodsky says.

Instead, a more disorganized and necrotic abscess developed, neutrophils failed to be activated, and the mice were less able to control the invading bacteria. These animals experienced higher levels of bacteria in their intestines and succumbed to their infections.

Groundwork for the future

The researchers believe the monocytes are responsible for recruiting neutrophils to the site of infection and thus launching the formation of the granuloma, helping to control the bacteria. This leading role for monocytes may exist beyond the intestines, the researchers believe.

We hypothesize that it’s a general role for the monocytes in other tissues as well.”

Igor Brodsky, senior author

But the discoveries also point to the intestines as a key site of engagement between the immune system and Yersinia.

“Previous to this study we knew of Peyer’s patches to be the primary site where the body interacts with the outside environment through the mucosal tissue of the intestines,” says Brodsky. Peyer’s patches are small areas of lymphoid tissue present in the intestines that serve to regulate the microbiome and fend off infection.

In future work, Brodsky and colleagues hope to continue to piece together the mechanism by which monocytes and neutrophils contain the bacteria, an effort they’re pursing in collaboration with Sunny Shin’s lab in the Perelman School of Medicine’s microbiology department.

A deeper understanding of the molecular pathways that regulate this immune response could one day offer inroads into host-directed immune therapies, by which a drug could tip the scales in favor of the host immune system, unleashing its might to fully eradicate the bacteria rather than simply corralling them in granulomas.

“These therapies have caused an explosion of excitement in the cancer field,” Brodsky says, “the idea of reinvigorating the immune system. Conceptually we can also think about how to coax the immune system to be reinvigorated to attack pathogens in these settings of chronic infection as well.”

Source:

University of Pennsylvania

Journal reference:

Sorobetea, D., et al. (2023). Inflammatory monocytes promote granuloma control of Yersinia infection. Nature Microbiology. doi.org/10.1038/s41564-023-01338-6.

Microbiology

Wayne State team discovers a simple technology to detect active TB infection antibodies

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Reviewed by Emily Henderson, B.Sc.Mar 13 2023

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-specific transketolase (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. 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.”

Lobelia Samavati, M.D., Professor, Center for Molecular Medicine and Genetics, School of Medicine, Wayne State University

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.

Source:

Wayne State University

Journal reference:

Talreja, J., et al. (2023). Discovery of Novel Transketolase Epitopes and the Development of IgG-Based Tuberculosis Serodiagnostics. Microbiology Spectrum. doi.org/10.1128/spectrum.03377-22.

Microbiology

Killing Even Antibiotic-Resistant Bacteria: A New Infection-Fighting Wound Spray

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The World Health Organization (WHO) has listed antibiotic resistance as a top ten global health threat, making it imperative to find new ways to combat resistant bacteria and decrease the reliance on antibiotics. In response to this pressing issue, researchers at the Chalmers University of Technology in Sweden have developed a new spray that has the ability to kill even antibiotic-resistant bacteria. This innovative solution can be used for wound care and directly on medical devices such as implants.

“Our innovation can have a dual impact in the fight against antibiotic resistance. The material has been shown to be effective against many different types of bacteria, including those that are resistant to antibiotics, such as Methicillin-resistant Staphylococcus aureus (MRSA), while also having the potential to prevent infections and thus reduce the need for antibiotics,” says Martin Andersson, head of research for the study and professor at the Department of Chemistry and Chemical Engineering at Chalmers.

It is already estimated that antibiotic-resistant bacteria cause nearly 1.3 million deaths a year worldwide. As part of the effort to slow down the spread and development of drug resistance, researchers at Chalmers are developing a new antibacterial material that can be used in healthcare and become an effective tool to fight antibiotic resistance.

The material consists of small hydrogel particles equipped with a type of peptide that effectively kills and binds bacteria. Attaching the peptides to the particles provides a protective environment and increases the stability of the peptides. This allows them to work together with body fluids such as blood, which otherwise inactivates the peptides, making them difficult to use in healthcare. In previous studies, the researchers showed how the peptides can be used for wound care materials such as wound dressings.

They have now published two new studies in which the bactericidal material is used in the form of a wound spray and as a coating on medical devices that are introduced into our bodies. This new step in the research means that the innovation can be used in more ways and be of even greater benefit in healthcare.

The wound spray, which can reach into deep wounds and other open areas on the body where bacteria can enter, is flexible and very useful for treating and preventing infection. The new material has many advantages over existing sprays and disinfectants

“The substance in this wound spray is completely non-toxic and does not affect human cells. Unlike existing bactericidal sprays, it does not inhibit the body’s healing process. The materials, which are simply sprayed onto the wound, can also kill the bacteria in a shorter time,” says Edvin Blomstrand, an industrial doctoral student at the Department of Chemistry and Chemical Engineering at Chalmers University of Technology and one of the lead authors of the scientific article.

For treatments in which materials such as implants and catheters are inserted into our bodies, infections are a major problem. Therefore, there is a great need for new antibacterial biomaterials, i.e. materials that treat, replace or modify organs, tissue, or functions in a biological body. One of the major sources of hospital-acquired infection comes from the usage of urinary catheters. The Chalmers researchers’ new coating can now be an effective new tool for reducing this risk and preventing infections.

“Although the catheters are sterile when unpacked, they can become contaminated with bacteria while they are being introduced into the body, which can lead to infection. One major advantage of this coating is that the bacteria are killed as soon as they come into contact with the surface. Another is that it can be applied to existing products that are already used in healthcare, so it is not necessary to produce new ones,” says Annija Stepulane, a doctoral student at the Department of Chemistry and Chemical Engineering at Chalmers and one of the lead authors of the article.

In the study, the researchers tested the coating on silicone materials used for catheters, but they see opportunities to use it on other biomaterials.

The research on the antibacterial materials is being conducted in collaboration with the spin-off company Amferia AB, which is also commercializing the technology. Chalmers and Amferia have previously presented the antibacterial material in the form of hydrogel wound dressings, which are presently under clinical investigation for both human and animal wound care.

The beneficial properties of antimicrobial peptides have been known for many years. They exist in thousands of different variants in the natural immune systems of humans, animals, and plants, and researchers have long sought to mimic and harness the peptides to prevent and treat infections. In their natural state, these peptides are rapidly broken down when they come into contact with body fluids such as blood, which makes their direct clinical use difficult. In the materials the researchers are developing, they have solved this problem by binding the peptides to particles. For both the spray and the coating, they have been able to measure that the bactericidal effect of the materials lasts for up to 48 hours in contact with body fluids and as long as a few years without contact with body fluids.

The researchers have shown that 99.99 percent of bacteria are killed by the material and that the bactericidal capacity is active for approximately 48 hours, enabling its use in a wide range of clinical applications. Since the materials are non-toxic, they can be used directly on or in the body, preventing or curing an infection without adversely affecting the natural healing process.

References: “Cross-linked lyotropic liquid crystal particles functionalized with antimicrobial peptides” by Edvin Blomstrand, Anand K. Rajasekharan, Saba Atefyekta and Martin Andersson, 22 September 2022, International Journal of Pharmaceutics.
DOI: 10.1016/j.ijpharm.2022.122215

“Multifunctional Surface Modification of PDMS for Antibacterial Contact Killing and Drug-Delivery of Polar, Nonpolar, and Amphiphilic Drugs” by Annija Stepulane, Anand Kumar Rajasekharan and Martin Andersson, 2 November 2022, ASC Applied Bio Materials.
DOI: 10.1021/acsabm.2c00705

Chalmers University of Technology

Microbiology

New discoveries made regarding autism onset in mouse models

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Reviewed by Emily Henderson, B.Sc.Mar 10 2023

Although autism is a common neurodevelopmental disorder, the multiple factors behind its onset are still not fully understood. Animal models of idiopathic autism, especially mice, are often used to help researchers understand the complicated mechanisms behind the disorder, with BTBR/J being the most commonly used mouse model in the world.

Now, an international research collaboration including Kobe University’s Professor TAKUMI Toru and Researcher Chia-wen Lin et al. have made new discoveries regarding autism onset in mouse models.

In their detailed series of experiments and analyses of BTBR/J mice and the other subspecies BTBR/R, they revealed that endogenous retrovirus activation increases a fetus’s susceptibility to autism. They also discovered that BTBR/R exhibits autistic-like behaviors without reduced learning ability, making it a more accurate model of autism than the widely-used BTBR/J model.

It is hoped that further research will contribute towards better classification of autism types, as well as the creation of new treatment strategies for neurodevelopmental disorders.

These research results were published in Molecular Psychiatry on March 7, 2023

Main points

  • The researchers analyzed BTBR/J, a widely used mouse model of autism, and its subspecies BTBR/Rusing MRI. This revealed that the corpus callosum, which connects the left and right hemispheres of the brain, was impaired in BTBR/J mice but not in BTBR/R mice.
  • Genome and transcription analysis showed that BTBR mice have increased levels of endogenous retrovirus genes.
  • Furthermore, single-cell RNA analysis of BTBR/R mice revealed changes in the expression of various genes (including stress response genes) that are indicative of endogenous retrovirus activation.
  • Even though BTBR/J and BTBR/R mice have the same ancestry, the results of various behavioral analysis experiments revealed differences in spatial learning ability and other behaviors between the two types of model mice.

Research background

Autism (autism spectrum disorder) is a neurodevelopmental disorder that remains largely unexplored despite the rapidly increasing number of patients. Reasons for this continuing increase in people diagnosed with autism include changes to diagnostic criteria and older fathers becoming more common. Autism is strongly related to genetic factors and can be caused by abnormalities in DNA structure, such as copy number variations. Animal models, especially mice, are often used in research to illuminate the pathology of autism. Among these models, BTBR/J is a mouse model of the natural onset of autism that is commonly used. Studies have reported various abnormalities in BTBR/J mice including impairment of the corpus callosum (which connects the left and right hemispheres of the brain) and excessive immune system signaling. However, it is not fully understood why this particular lineage displays autistic-like behavioral abnormalities.

The aim of the current study was to shed light on the onset mechanism of these autistic-like behavioral abnormalities by conducting comparative analysis on BTBR/J and its subspecies BTBR/R.

Research findings

First of all, the researchers conducted MRI scans on BTBR/J and BTBR/R mice to investigate structural differences in each region of the brain. The results revealed that there were differences between BTBR/J and BTBR/R mice in 33 regions including the amygdala. A particularly prominent difference discovered was that even though BTBR/J’s corpus callosum is impaired, BTBR/R’s is normal.

Next, the research group used the array CGH method to compare BTBR/R’s copy number variations with that of a normal mouse model (B6). They revealed that BTBR/R mice had significantly increased levels of endogenous retroviruses (ERV) in comparison to B6 mice. Furthermore, qRT-PCR tests revealed that these retroviruses were activated in BTBR/R mice. On the other hand, in B6 mice there was no change in the expression of LINE ERV (which is classified in the same repetitive sequence), indicating that this retroviral activation is specific to BTBR.

Subsequently, the researchers carried out single-cell RNA analysis on the tissue of embryonic BTBR mice (on the AGM and yolk sac). The results provide evidence of ERV activation in BTBR mice, as expression changes were observed in a group of genes downstream of ERV.

Lastly, the researchers comprehensively investigated the differences between BTBR/J and BTBR/R on a behavioral level. BTBR/R mice were less anxious than BTBR/J and showed qualitative changes in ultrasound vocalizations, which are measured as a way to assess communicative ability in mice. BTBR/R mice also exhibited more self-grooming behaviors and buried more marbles in the marble burying test. These two tests were designed to detect repetitive behavioral abnormalities in autistic individuals. From the results, it was clear that BTBR/R exhibits more repetitive behaviors (i.e. it is more symptomatic) than BTBR/J. The 3-chamber social interaction test, which measures how closely a mouse will approach another mouse, also revealed more pronounced social deficits in BTBR/R than BTBR/J mice (Figure 4i). In addition, a Barnes maze was used to conduct a spatial learning test, in which BTBR/J mice exhibited reduced learning ability compared to B6 (normal mice). BTBR/R mice, on the other hand, exhibited similar ability to B6.

Overall, the study revealed that retrovirus activation causes the copy number variants in BTBR mice to increase, which leads to the differences in behavior and brain structure seen in BTBR/J and BTBR/R mice (Figure 5).

Further developments

BTBR/J mice are widely used by researchers as a mouse model of autism. However, the results of this study highlight the usefulness of the other lineage of BTBR/R mice because they exhibit autistic-like behavior without compromised spatial learning ability. The results also suggest that it may be possible to develop new treatments for autism that suppress ERV activation. Furthermore, it is necessary to classify autism subtypes according to their onset mechanism, which is a vital first step towards opening up new avenues of treatment for autism.

Source:

Kobe University

Journal reference:

Lin, C-W., et al. (2023) An old model with new insights: endogenous retroviruses drive the evolvement toward ASD susceptibility and hijack transcription machinery during development. Molecular Psychiatry. doi.org/10.1038/s41380-023-01999-z.

Microbiology

Clinical trial shows safety and immunogenicity of temperature-stable experimental TB vaccine

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Reviewed by Emily Henderson, B.Sc.Mar 6 2023

A clinical trial testing a freeze-dried, temperature-stable experimental tuberculosis (TB) vaccine in healthy adults found that it was safe and stimulated both antibodies and responses from the cellular arm of the immune system. The Phase 1 trial was supported by the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health. A non-temperature stable form of the candidate previously had been tested in several clinical trials. However, this was the first clinical trial of any subunit TB vaccine candidate in a temperature-stable (thermostable) form. Results are published in Nature Communications.

The experimental vaccine, ID93+GLA-SE, was developed by Christopher B. Fox, Ph.D., and scientists at the Access to Advanced Health Institute (formerly the Infectious Disease Research Institute) in Seattle. It is a recombinant subunit vaccine made from four proteins of Mycobacterium tuberculosis bacteria combined with GLA-SE, an immune-stimulating adjuvant. The freeze-dried formulation does not require refrigeration and is mixed with sterile water just prior to injection. Thermostable vaccines are desirable in settings where maintaining cold or frozen vaccines for long periods can be costly and difficult.

The current trial investigated whether administering temperature-stable vaccine containing both ID93 and GLA-SE in a single vial would be as effective at inducing an immune response as a regimen in which non-thermostable ID93 and liquid GLA-SE are held in two vials and combined prior to injection. A single-vial presentation of a thermostable vaccine would have clear advantages in ease of storage, transport and administration, the investigators note.

Daniel F. Hoft, M.D., Ph.D., director of the Saint Louis University Center for Vaccine Development, led the single-site trial at the university’s School of Medicine. Twenty-three participants received the thermostable single-vial regimen, while 22 participants received the two-vial, non-thermostable regimen. Both vaccine presentations were safe and well-tolerated. Recipients of the single-vialled thermostable vaccine had robust T-cell responses and produced higher levels of antibodies in the blood than those receiving the non-thermostable two-vial presentation.

The investigators note some limitations in this small trial. For example, no established correlates of protection define what immune responses are required for vaccine-induced protection from TB disease. Therefore, it is not possible to say whether the enhanced immune responses seen in the thermostable vaccine formulation would translate to improved protective vaccine efficacy. Nevertheless, they conclude, results of this trial demonstrate “a proof-of-concept that adjuvant-containing vaccines can be formulated in a freeze-dried single-vial presentation without detrimentally impacting clinical immunogenicity or safety characteristics.”

Source:

National Institutes of Health

Journal reference:

Sagawa, Z.K., et al. (2023) Safety and immunogenicity of a thermostable formulation of the ID93 + GLA-SE tuberculosis vaccine candidate in healthy adults. Nature Communications. doi.org/10.1038/s41467-023-36789-2.