Tag Archives: Virus

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

Low-cost, universal oral COVID-19 vaccine prevents severe respiratory illness in hamsters

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

A UCLA-led team has developed an inexpensive, universal oral COVID-19 vaccine that prevented severe respiratory illness and weight loss when tested in hamsters, which are naturally susceptible to SARS-CoV-2. It proved as effective as vaccines administered by injection or intranasally in the research.

If ultimately approved for human use, it could be a weapon against all COVID-19 variants and boost uptake, particularly in low- and middle-income countries, and among those with an aversion to needles.

The study is published in the peer-reviewed journal Microbiology Spectrum.

The oral vaccine is based primarily on the nucleocapsid protein, which is the most abundantly expressed of the virus’s four major structural proteins and evolves at a much slower rate than the frequently mutating spike protein. The vaccine utilizes a highly weakened bacterium to produce the nucleocapsid protein in infected cells as well as the membrane protein, which is another highly abundant viral structural protein.

Being a universal vaccine based primarily upon the nucleocapsid protein, the vaccine is resistant to the incessant mutations of the SARS-CoV-2 spike protein upon which virtually all current vaccines are based. As a result, current vaccines rapidly become obsolete, requiring that they repeatedly be re-engineered. Hence, our vaccine should protect against new and emerging variants of SARS-CoV-2.”

Dr. Marcus Horwitz, senior author, distinguished professor of medicine in the Division of Infectious Diseases and of microbiology, immunology and molecular genetics at the David Geffen School of Medicine at UCLA

Oral delivery also makes it easier to distribute the vaccine in resource poor areas of the world by eliminating the need for needles, syringes, and trained personnel to deliver injectable vaccines, he added. “An oral vaccine may also be attractive to many people with vaccine hesitancy on account of fear of needles.”

The researchers noted that while it worked exceptionally well in preventing severe respiratory illness, it did not provide full protection against high viral loads in the hamsters. Also, they did not test it against the Omicron strain, which contains a nearly identical nucleocapsid protein, because of this strain’s low virulence in the golden Syrian hamsters they used.

But the vaccine, they write, “is efficacious when administered via the oral route against COVID-19-like disease in a highly demanding animal model. This conveniently administered, easily manufactured, inexpensive, and readily stored and transported vaccine could play a major role in ending the COVID-19 pandemic by protecting immunized individuals from serious disease from current and future strains of SARS-CoV-2.”

The next step in the process will be to manufacture the vaccine for oral administration via an acid-resistant enteric capsule that will allow the vaccine to be safely released in the small intestine, Horwitz said. It will then be tested for safety, immunogenicity, and efficacy in humans.

“We also plan to expand the vaccine to protect against infections caused by other types of potentially pandemic coronaviruses such as the virus that causes Middle Eastern Respiratory Syndrome (MERS),” he added.

Additional authors are Qingmei Jia and Saša Masleša-Galić of UCLA; Helle Bielefeldt-Ohmann of the University of Queensland, Australia; and Rachel Maison, Airn Hartwig, and Richard Bowen of Colorado State University.

This study was supported by a Corona Virus Seed grant from the UCLA AIDS Institute and Charity Treks and by the National Institutes of Health (AI141390).

Source:

UCLA Health

Journal reference:

Jia, Q., et al. (2023). Oral Administration of Universal Bacterium-Vectored Nucleocapsid-Expressing COVID-19 Vaccine is Efficacious in Hamsters. Microbiology Spectrum. doi.org/10.1128/spectrum.05035-22.

Microbiology

Co-infection with MRSA ‘superbug’ could make COVID-19 outcomes even more deadly

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

Global data shows nearly 10 per cent of severe COVID-19 cases involve a secondary bacterial co-infection – with Staphylococcus aureus, also known as Staph A., being the most common organism responsible for co-existing infections with SARS-CoV-2. Researchers at Western have found if you add a ‘superbug’ – methicillin-resistant Staphylococcus aureus (MRSA) – into the mix, the COVID-19 outcome could be even more deadly.

The mystery of how and why these two pathogens, when combined, contribute to the severity of the disease remains unsolved. However, a team of Western researchers has made significant progress toward solving this “whodunit”.

New research by Mariya Goncheva, Richard M. Gibson, Ainslie C. Shouldice, Jimmy D. Dikeakos and David E. Heinrichs, has revealed that IsdA, a protein found in all strains of Staph A., enhanced SARS-CoV-2 replication by 10- to 15-fold. The findings of this study are significant and could help inform the development of new therapeutic approaches for COVID-19 patients with bacterial co-infections.

Interestingly, the study, which was recently published in iScience, also showed that SARS-CoV-2 did not affect the bacteria’s growth. This was contrary to what the researchers had initially expected.

We started with an assumption that SARS-CoV-2 and hospitalization due to COVID-19 possibly caused patients to be more susceptible to bacterial infections which eventually resulted in worse outcomes.”

Mariya Goncheva

Goncheva is a former postdoctoral associate, previously with the department of microbiology and immunology at Schulich School of Medicine & Dentistry.

Goncheva said bacterial infections are most commonly acquired in hospital settings and hospitalization increases the risk of co-infection. “Bacterial infections are one of the most significant complications of respiratory viral infections such as COVID-19 and Influenza A. Despite the use of antibiotics, 25 per cent of patients co-infected with SARS-CoV-2 and bacteria, die as a result. This is especially true for patients who are hospitalized, and even more so for those in intensive care units. We were interested in finding why this happens,” said Goncheva, lead investigator of the study.

Goncheva, currently Canada Research Chair in virology and professor of biochemistry and microbiology at the University of Victoria, studied the pathogenesis of multi-drug resistant bacteria (such as MRSA) supervised by Heinrichs, professor of microbiology and immunology at Schulich Medicine & Dentistry.

When the COVID-19 pandemic hit, she pivoted to study interactions between MRSA and SARS-CoV-2.

For this study, conducted at Western’s level 3 biocontainment lab, Imaging Pathogens for Knowledge Translation (ImPaKT), Goncheva’s work created an out-of-organism laboratory model to study the interactions between SARS-CoV-2 and MRSA, a difficult-to-treat multi-drug resistant bacteria.

“At the beginning of the pandemic, the then newly opened ImPaKT facility made it possible for us to study the interactions between live SARS-CoV-2 virus and MRSA. We were able to get these insights into molecular-level interactions due to the technology at ImPaKT,” said Heinrichs, whose lab focuses on MRSA and finding drugs to treat MRSA infections. “The next step would be to replicate this study in relevant animal models.”

Source:

University of Western Ontario

Journal reference:

Goncheva, M. I., et al. (2023). The Staphylococcus aureus protein IsdA increases SARS CoV-2 replication by modulating JAK-STAT signaling. IScience. doi.org/10.1016/j.isci.2023.105975.

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

New SARS-CoV-2 Omicron XBB.1.5 variant has high transmissibility and infectivity, study finds

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Mar 20 2023Reviewed by Danielle Ellis, B.Sc.

COVID-19 has caused significant global panic after its rapid emergence more than 3 years ago. Although we now have highly effective vaccines against the SARS-CoV-2 virus, which causes COVID-19, scientists continue to study emerging SARS-CoV-2 variants in order to safeguard public health and devise global preventive strategies against emerging variants. A team led by Japanese researchers has recently discovered that the SARS-CoV-2 Omicron XBB.1.5 variant, prevalent in the Western hemisphere, has high transmissibility and infectivity.

New SARS-CoV-2 Omicron XBB.1.5 variant has high transmissibility and infectivity, study finds
New SARS-CoV-2 variant may jeopardize public health across the globe. The SARS-CoV-2 Omicron XBB.1.5 variant spreads rapidly and is more infectious than its historic precursor. Image Credit: The University of Tokyo

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for millions of deaths worldwide. Although scientists have designed novel vaccines to counter COVID-19, they are constantly on the lookout for emerging variants that can bypass vaccine resistance and potentially jeopardize global public health. A team led by Japanese researchers has recently been successful in characterizing the new SARS-CoV-2 Omicron XBB.1.5 variant, which was first detected in October 2022. Their findings were published on January 31, 2023 in volume 23 of The Lancet Infectious Diseases.​​​

Says senior author Prof. Kei Sato from the Division of Systems Virology, The Institute of Medical Science, The University of Tokyo, Japan, “Because the Omicron XBB.1.5 variant can spread more rapidly than previous variants and has a potential to cause the next epidemic surge, we should carefully monitor it to safeguard public health.”

While studying emerging variants of the SARs-CoV-2 Omicron lineage, the research team made a startling discovery: the SARS-CoV-2 Omicron XBB.1.5 variant has a novel mutation in the spike (S) protein—the protein that anchors the virus firmly to the human angiotensin converting enzyme-2 (ACE2) receptor, thus facilitating the invasion of human cells. The serine-to-proline amino acid mutation noted at residue no. 486 in the S protein is virologically concerning because of a variety of reasons.

Sharing his concerns, first author Keiya Uriu from the Division of Systems Virology, Department of Microbiology and Immunology, The University of Tokyo, Japan, says, “In late 2022, the SARS-CoV-2 Omicron BQ.1 and XBB lineages, characterized by amino acid substitutions in the S protein and increased viral fitness, had become predominant in the Western and Eastern Hemisphere, respectively. In 2022, we elucidated the characteristics of a variety of newly emerging SARS-CoV-2 Omicron subvariants. At the end of 2022, the XBB.1.5 variant, a descendant of XBB.1 that acquired the S:S486P substitution, emerged and was rapidly spreading in the USA.”

To gain mechanistic insights into the infectivity, transmissibility, and immune response associated with XBB.1.5, the team conducted a series of experiments. For instance, upon conducting epidemic dynamics analysis—statistical modeling that facilitates the analysis of the general characteristics of any epidemic—the team realized that the relative effective reproduction number (Re) of XBB.1.5 was 1.2-fold greater than that of the parental XBB.1. This indicated that an individual with the XBB.1.5 variant could infect 1.2 times more people in the population than someone with the parental XBB.1 variant. Moreover, the team also realized that, as of December 2022, XBB.1.5 was rapidly outcompeting BQ.1.1, the predominant lineage in the United States.

Co-first-author Jumpei Ito from the Division of Systems Virology, remarks, “Our data suggest that XBB.1.5 will rapidly spread worldwide in the near future.”

The team also studied the virological features of XBB.1.5 to determine how tightly the S protein of the new variant interacts with the human ACE2 receptor. To this end, the researchers conducted a yeast surface display assay. The results showed that the dissociation constant (KD) corresponding to the physical interaction between the XBB.1.5 S receptor-binding domain (RBD) and the human ACE2 receptor is significantly (4.3-fold) lower than that for XBB.1 S RBD. “In other words, the XBB.1.5 variant binds to human ACE2 receptor with very high affinity,” explains Shigeru Fujita from the Division of Systems Virology.

Further experiments using lentivirus-based pseudoviruses also showed that XBB.1.5 had approximately 3-fold higher infectivity than XBB.1. These results suggest that XBB.1.5 exhibits a remarkably strong affinity to the human ACE2 receptor, which can be attributed to the S486P substitution.

The study by Prof. Sato and his team led to another important discovery from an immunization perspective. The XBB.1.5 S protein was found to be highly resistant to neutralization antibodies elicited by breakthrough infection with the BA.2/BA.5 subvariants. In other words, patients with prior infection from the BA.2/BA.5 subvariants may not show robust immunity against XBB.1.5, increasing their chances of infection and disease.

The results of our virological experiments explain why the Omicron XBB.1.5 variant has a higher transmissibility than past variants: This variant acquired strong binding ability to human ACE2 while maintaining a higher ability to escape from neutralizing antibodies.”

​​​​​​​Yusuke Kosugi, Division of Systems Virology, Department of Microbiology and Immunology, The University of Tokyo, Japan

Contributing members of The Genotype to Phenotype Japan (G2P-Japan) Consortium conclude, “The SARS-CoV-2 Omicron XBB.1.5 variant does show enhanced transmissibility. Although few cases have been detected in the Eastern hemisphere, it could become a looming threat. Imminent prevention measures are needed.”

​​​​​​​Thanks to the research team for the early warning! Meanwhile, we must continue adopting safe practices to defend ourselves from XBB.1.5. 

Source:

The University of Tokyo

Journal reference:

Uriu, K., et al. (2023) Enhanced transmissibility, infectivity, and immune resistance of the SARS-CoV-2 omicron XBB.1.5 variant. The Lancet Infectious Diseases. doi.org/10.1016/S1473-3099(23)00051-8.

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

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

    The ‘Rapunzel’ virus: an evolutionary oddity

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    A recent study in the Journal of Biological Chemistry has revealed the secret behind an evolutionary marvel: a bacteriophage with an extremely long tail. This extraordinary tail is part of a bacteriophage that lives in inhospitable hot springs and preys on some of the toughest bacteria on the planet.

    Bacteriophages are a group of viruses that infect and replicate in bacteria and are the most common and diverse things on Earth.

    “Bacteriophages, or phages for short, are everywhere that bacteria are, including the dirt and water around you and in your own body’s microbial ecosystem as well,” said Emily Agnello, a graduate student at the University of Massachusetts Chan Medical School and the lead author on the study.

    Unlike many of the viruses that infect humans and animals that contain only one compartment, phages consist of a tail attached to a spiky, prismlike protein shell that contains their DNA.

    Phage tails, like hairstyles, vary in length and style; some are long and bouncy while others are short and stiff. While most phages have short, microscopic tails, the “Rapunzel bacteriophage” P74-26 has a tail 10 times longer than most and is nearly 1 micrometer long, about the width of some spider’s silk. The “Rapunzel” moniker is derived from the fairy tale in which a girl with extremely long hair was locked in a tower by an evil witch.

    Brian Kelch, an associate professor of biochemistry and molecular biotechnology at UMass Chan who supervised the work, described P74-26 as having a “monster of a tail.”

    Phage tails are important for puncturing bacteria, which are coated in a dense, viscous substance. P74-26’s long tail allows it to invade and infect the toughest bacteria. Not only does P74-26 have an extremely long tail, but it is also the most stable phage, allowing it to exist in and infect bacteria that live in hot springs that can reach over 170° F. Researchers have been studying P74-26 to find out why and how it can exist in such extreme environments.

    To work with a phage that thrives in such high temperatures, Agnello had to adjust the conditions of her experiments to coax the phage tail to assemble itself in a test tube. Kelch said Agnello created a system with which she could induce rapid tail self-assembly.

    “Each phage tail is made up of many small building blocks that come together to form a long tube. Our research finds that these building blocks can change shape, or conformation, as they come together,” Agnello said. “This shape-changing behavior is important in allowing the building blocks to fit together and form the correct structure of the tail tube.”

    The researchers used high-power imaging techniques as well as computer simulations and found that the building blocks of the tail lean on each other to stabilize themselves.

    “We used a technique called cryo-electron microscopy, which is a huge microscope that allows us to take thousands of images and short movies at a very high magnification,” Agnello explained. “By taking lots of pictures of the phage’s tail tubes and stacking them together, we were able to figure out exactly how the building blocks fit together.”

    They found P74-26 uses a “ball and socket” mechanism to sturdy itself. In addition, the tail is formed from vertically stacking rings of molecules that make a hollow canal.

    “I like to think about these phage building blocks as kind of like Legos,” Kelch said. “The Lego has studs on one side and the holes or sockets on the other.”

    He added: “Imagine a Lego where the sockets start off closed. But as you start to build with the Legos, the sockets begin to open up to allow the studs on other Legos to build a larger assembly. This movement is an important way that these phage building blocks self-regulate their assembly.”

    Kelch pointed out that, compared with most phages, P74-26 uses half the number of building blocks to form stacking rings that make up the tail.

    “We think what has happened is that some ancient virus fused its building blocks into one protein. Imagine two small Lego bricks are fused into one large brick with no seams. This long tail is built with larger, sturdier building blocks,” Kelch explained. “We think that could be stabilizing the tail at high temperatures.”

    The researchers now plan to use genetic manipulation to alter the length of the phage tail and see how that changes its behavior.

    Phages occupy almost every corner of the globe and are important to a variety of industries like healthcare, environmental conservation and food safety. In fact, long-tailed phages like P74-26 have been used in preliminary clinical trials to treat certain bacterial infections.

    “Bacteriophages are gaining ever-growing interest as an alternative to antibiotics for treating bacterial infections,” Agnello said. “By studying phage assembly, we can better understand how these viruses interact with bacteria, which could lead to the development of more effective phage-based therapies. … I believe that studying unique, interesting things can lead to findings and applications that we can’t even yet imagine.”

  • Emily Agnello, Joshua Pajak, Xingchen Liu, Brian A. Kelch. Conformational dynamics control assembly of an extremely long bacteriophage tail tube. Journal of Biological Chemistry, 2023; 103021 DOI: 10.1016/j.jbc.2023.103021

    • American Society for Biochemistry and Molecular Biology
    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