Tag Archives: Peptides

Microbiology

First-in-human nanoparticle HIV vaccine induces broad and publicly targeted helper T cell responses

Leave a comment

if (g_displayableSlots.mobileTopLeaderboard) {
pushDisplayAd(function() { googletag.display(‘div-gpt-mobile-top-leaderboard’); });
}

Reviewed by Lily Ramsey, LLMMay 25 2023

Researchers from Fred Hutchinson Cancer Center in Seattle, Scripps Research in La Jolla, California, IAVI and other collaborating institutions have characterized robust T-cell responses in volunteers participating in the IAVI G001 Phase 1 clinical trial to test the safety and immune response of a self-assembling nanoparticle HIV vaccine.

Their work, published in Science Translational Medicine, signals a major step toward development of a vaccine approach to end the HIV/AIDS epidemic worldwide. The antigen used in this study was jointly developed by IAVI and Scripps Research and has been shown in previous analyses to stimulate VRC01-class B cells, an immune response considered promising enough for boosting in further studies.

We were quite impressed that this vaccine candidate produced such a vigorous T-cell response in almost all trial participants who received the vaccine. These results highlight the potential of this HIV-1 nanoparticle vaccine approach to induce the critical T-cell help needed for maturing antibodies toward the pathway of broadly neutralizing against HIV.”

Julie McElrath, MD, PhD, senior vice president and director of Fred Hutch’s Vaccine and Infectious Disease Division and co-senior author of the study

However, she added, this is the first step, and heterologous booster vaccines will still be needed to eventually produce VRC01-class broadly neutralizing antibodies, which in previous studies have demonstrated the ability to neutralize approximately 90% of HIV strains.

“We showed previously that this vaccine induced the desired B-cell responses from HIV broadly neutralizing antibody precursors. Here we demonstrated strong CD4 T-cell responses, and we went beyond what is normally done by drilling down to identify the T cell epitopes and found several broadly immunogenic epitopes that might be useful for developing boosters and for other vaccines,” William Schief, PhD, executive director of vaccine design for IAVI’s Neutralizing Antibody Center at Scripps Research and professor, Department of Immunology and Microbiology, at Scripps Research, who is co-senior author of the study.

if (g_displayableSlots.mobileMiddleMrec) {
pushDisplayAd(function() { googletag.display(‘div-gpt-mobile-middle-mrec’); });
}

The trial is a phase 1, randomized, double-blind and placebo-controlled study to evaluate the safety and effectiveness of a nanoparticle HIV vaccine in healthy adult volunteers without HIV. It was comprised of two groups with 18 vaccine and six placebo recipients per group, with 48 total enrollees. Participants were given two doses of the vaccine or placebo eight weeks apart.

McElrath acknowledged the groundbreaking work of her lab team, the biostatistical team and Fred Hutch’s Vaccine Trials Unit for their invaluable contributions to the study. The Vaccine Trials Unit conducts multiple vaccine trials and was one of only two sites for this study.

Findings from the study include:

  • Vaccine-specific CD4 T cells were induced in almost all vaccine recipients.
  • Lymph node GC T follicular helper cells increased after vaccination compared to placebo.
  • Lumazine synthase protein, needed for self-assembly of the particle, also induced T-cell responses that can provide additional help to ultimately enhance efficacy in a sequential vaccine strategy.
  • Vaccine-specific CD4 T cells were polyfunctional and had diverse phenotypes.
  • LumSyn-specific CD8 T cells were highly polyfunctional and had a predominantly effector memory phenotype.
  • CD4 T-cell responses were driven by immunodominant epitopes with diverse and promiscuous HLA restriction.
  • CD8 T-cell responses to LumSyn were driven by HLA-A*02-restricted immunodominant epitopes B- and T-cell responses correlated within but not between LN and peripheral blood compartments.

This study was funded by the Bill & Melinda Gates Foundation Collaboration for AIDS Vaccine Discovery; IAVI Neutralizing Antibody Center; National Institute of Allergy and Infectious Diseases; and Ragon Institute of MGH, MIT and Harvard.

Study authors WRS and SM are inventors on a patent filed by Scripps and IAVI on the eOD-GT8 monomer and 60-mer immunogens (patent number 11248027, “Engineered outer domain (eOD) of HIV gp 120 and mutants thereof”). WRS, KWC and MJM are inventors on patents filed by Scripps, IAVI and Fred Hutch on immunodominant peptides from LumSyn (Title: Immunogenic compositions; filing no. 63127975).

if (g_displayableSlots.mobileBottomLeaderboard) {
pushDisplayAd(function() { googletag.display(‘div-gpt-mobile-bottom-leaderboard’); });
}

Source:

Fred Hutchinson Cancer Center

Journal reference:

Cohen, K. W., et al. (2023) A first-in-human germline-targeting HIV nanoparticle vaccine induced broad and publicly targeted helper T cell responses. Science Translational Medicine. doi.org/10.1126/scitranslmed.adf3309.

Microbiology

Novel computational platform can expand the pool of cancer immunotherapy targets

Leave a comment

Reviewed by Megan Craig, M.Sc.May 17 2023

Researchers at Children’s Hospital of Philadelphia (CHOP) and the University of California, Los Angeles (UCLA) have developed a computational platform capable of discovering tumor antigens derived from alternative RNA splicing, expanding the pool of cancer immunotherapy targets. The tool, called “Isoform peptides from RNA splicing for Immunotherapy target Screening” (IRIS), was described in a paper published today in the Proceedings of the National Academy of Sciences.

Immunotherapy has revolutionized cancer treatment, but for many cancers including pediatric cancers, the repertoire of antigens is incomplete, underscoring a need to expand the inventory of actionable immunotherapy targets. We know that aberrant alternative RNA splicing is widespread in cancer and generates a range of potential immunotherapy targets. In our study, we were able to show that our computational platform was able to identify immunotherapy targets that arise from alternative splicing, introducing a broadly applicable framework for discovering novel cancer immunotherapy targets that arise from this process.”

Yi Xing, PhD, co-senior author, director of the Center for Computational and Genomic Medicine at CHOP

Cancer immunotherapy has ushered in a sea change in the treatment of many hematologic cancers, harnessing the power of a patient’s own immune system to fight the disease. Chimeric antigen receptor T-cell (CAR-T) and T cell receptor-engineered T cell (TCR-T) therapies modify a patient’s own T cells to attack known antigens on the surface of cancer cells and have often led to durable responses for cancers that were once considered incurable. However, the field has encountered challenges in the solid tumor space, in large part due to a lack of known and suitable targets for these cancers, highlighting the need for novel approaches to expand the pool of immunotherapy targets.

Alternative splicing is an essential process that allows for one gene to code for many gene products, based on where the RNA is cut and joined, or spliced, before being translated into proteins. However, the splicing process is dysregulated in cancer cells, which often take advantage of this process to produce proteins that promote growth and survival, allowing them to replicate uncontrollably and metastasize. This happens in many adult and pediatric cancers. Scientists have suggested splicing dysregulation could be a source of novel tumor antigens for immunotherapy, but identifying such antigens has been a challenge.

To address this difficulty, the researchers created IRIS to leverage large-scale tumor and normal RNA sequencing data and incorporate multiple screening approaches to discover tumor antigens that arise due to alternative splicing. Integrating RNA sequencing-based transcriptomics data and mass spectrometry-based proteomics data, the researchers showed that hundreds of IRIS-predicted TCR targets are presented by human leukocyte antigen (HLA) molecules, the part of the human immune system that presents antigens to T cells.

if (g_displayableSlots.mobileMiddleMrec) {
pushDisplayAd(function() { googletag.display(‘div-gpt-mobile-middle-mrec’); });
}

The researchers then applied IRIS to RNA sequencing data from neuroendocrine prostate cancer (NEPC), a metastatic and highly lethal disease known to involve shifts in RNA splicing, as discovered in a prior study by CHOP and UCLA researchers. From 2,939 alternative splicing events enriched in NEPC, IRIS predicted 1,651 peptides as potential TCR targets. The researchers then applied a more stringent screening test, which prioritized 48 potential targets. Interestingly, the researchers found that these targets were highly enriched for peptides encoded by short sequences of less than 30 nucleotides in length – also known as “microexons” – which may arise from a unique program of splicing dysregulation in this type of cancer.

To validate the immunogenicity of these targets, the researchers isolated T cells reactive to IRIS-predicted targets, and then used single-cell sequencing to identify the TCR sequences. The researchers modified human peripheral blood mononuclear cells with seven TCRs and found they were highly reactive against targets predicted by IRIS to be good immunotherapy candidates. One TCR was particularly efficient at killing tumor cells expressing the target peptide of interest.

“Immunotherapy is a powerful tool that has had a significant impact on the treatment of some cancers, but the benefits have not been fully realized in many lethal cancers that could benefit from this approach,” said Owen N. Witte, MD, University Professor of Microbiology, Immunology, and Molecular Genetics and member of the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA. “The discovery of new antigenic targets that may be shared among different patients – and even different tumor types – could be instrumental in expanding the value of cell-based therapies. Analyzing massive amounts of data on tumor and normal tissues, which requires sophisticated computational tools like those developed by the Xing Lab, provides actionable insights on targets that one day could be tested in the clinic.”

“This proof-of-concept study demonstrates that alternatively spliced RNA transcripts are viable targets for cancer immunotherapy and provides a big data and multiomics-powered computational platform for finding these targets,” Dr. Xing added. “We are applying IRIS for target discovery across a wide range of pediatric and adult cancers. We are also developing a next-generation IRIS platform that harnesses newer transcriptomics technologies, such as long read and single cell analysis.”

This research was supported in part by the Immuno-Oncology Translational Network (IOTN) of the National Cancer Institute’s Cancer Moonshot Initiative, other National Institutes of Health funding, the Parker Institute for Cancer Immunotherapy, the Cancer Research Institute, and the Ressler Family Fund.

Source:

Children’s Hospital of Philadelphia

Journal reference:

Pan, Y., et al. (2023) IRIS: Discovery of cancer immunotherapy targets arising from pre-mRNA alternative splicing. PNAS. doi.org/10.1073/pnas.2221116120.

Microbiology

New compound with antibacterial activity shows promising results within one hour in laboratory trials

Leave a comment

Reviewed by Emily Henderson, B.Sc.May 15 2023

Resistance to antibiotics is a problem that alarms the medical and scientific community. Bacteria resistant to three different classes of antibiotics, known as multi-drug resistant (MDR) bacteria, are far from rare. Some are even resistant to all currently available treatments and are known as pan-drug resistant (PDR). They are associated with dangerous infections and listed by the World Health Organization (WHO) as priority pathogens for drug development with maximum urgency.

An article published in a special issue of the journal Antibiotics highlights a compound with antibacterial activity that presented promising results within one hour in laboratory trials.

The study was led by Ilana Camargo, last author of the article, and conducted during the doctoral research of first author Gabriela Righetto at the Molecular Epidemiology and Microbiology Laboratory (LEMiMo) of the University of São Paulo’s São Carlos Institute of Physics (IFSC-USP) in Brazil.

The compound we discovered is a new peptide, Pln149-PEP20, with a molecular framework designed to enhance its antimicrobial activity and with low toxicity. The results can be considered promising insofar as the trials involved pathogenic bacteria associated with MDR infections worldwide.”

Adriano Andricopulo, co-author of the article

Although novel antibacterial drugs are urgently needed, the pharmaceutical industry is notoriously uninterested in pursuing them, mainly because research in this field is time-consuming and costly, requiring very long lead times to bring viable active compounds to market.

The Center for Innovation in Biodiversity and Drug Discovery (CIBFar), a Research, Innovation and Dissemination Center (RIDC) set up and funded by FAPESP, looks for molecules that can be used to combat multidrug-resistant bacteria.

Camargo and Andricopulo are researchers at CIBFar, as are two other co-authors who study promising bactericidal compounds: Leila Beltramini and José Luiz Lopes.

For over a decade, the group formed by the collaboration between Beltramini and Lopes has analyzed Plantaricin 149 and its analogs. Plantaricins are substances produced by the bacterium Lactobacillus plantarum to combat other bacteria.

if (g_displayableSlots.mobileMiddleMrec) {
pushDisplayAd(function() { googletag.display(‘div-gpt-mobile-middle-mrec’); });
}

Lactobacillus plantarum is commonly found in nature, especially in anaerobic plant matter, and in many fermented vegetable, meat and dairy products.

In the case of Plantaricin 149, Japanese researchers were the first to report its bactericidal action (in 1994) and since then scientists have been interested in obtaining more efficient synthetic analogs (molecules with small structural differences). In 2007, one of the first projects completed by the CIBFar team showed that the peptide inhibits pathogenic bacteria such as Listeria spp. and Staphylococcus spp. They then began studying synthetic analogs with stronger bactericidal activity than the original (causing more damage to the membrane of the combated microorganisms).

With the support of a scholarship from FAPESP, Righetto synthesized 20 analogs of Plantaricin 149, finding that Pln149-PEP20 had the best results so far and was also half the size of the original peptide. “The main advances in our research consist of the development of this smaller, more active and less toxic molecule, and the characterization of its action and propensity to develop resistance. It has proven to be highly promising in vitro – active against MDR bacteria and extensively resistant bacteria,” said Camargo, principal investigator for the project.

LEMiMo, the laboratory where the studies were conducted, has experience in characterizing bacterial isolates involved in outbreaks of hospital infections and holds a collection of bacteria selected for these trials in search of novel active compounds. The bacteria have the resistance profiles currently of greatest concern and were isolated during hospital outbreaks.

They are known in the scientific community by the term ESKAPE, an acronym for the scientific names of six highly virulent and antibiotic-resistant bacterial pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.

Further research can now be conducted to investigate the molecule’s action mechanism in more depth, to look for formulations, and possibly to develop an application. “In terms of the action mechanism, it’s also possible to use the cell morphology of the bacteria to identify cellular pathways affected by the peptide,” Righetto said. “As for optimization, the molecule can be functionalized by being linked to macrostructures, and the amino acid sequence can be modified.” Research is also needed on its cytotoxicity and on its selectivity (whether it affects healthy cells).

“We’re living in times of major global public health hazards due to a lack of antimicrobials that can be used to treat infections caused by extremely resistant bacteria. Antimicrobial peptides are targets of great interest for the development of novel candidate drugs. This novel molecule has the potential to be used as an innovative antimicrobial therapy, but further modifications and molecular optimizations still need to be investigated,” Andricopulo said.

Publication of the article also involved Harvard Medical School’s Infectious Disease Institute in Boston (USA) via researchers Paulo José Martins Bispo and Camille André.

Source:

São Paulo Research Foundation (FAPESP)

Journal reference:

Righetto, G. M., et al. (2023). Antimicrobial Activity of an Fmoc-Plantaricin 149 Derivative Peptide against Multidrug-Resistant Bacteria. doi.org/10.3390/antibiotics12020391.

Microbiology

Diet has a much stronger impact on intestinal microbiota than defensins

Leave a comment

Reviewed by Emily Henderson, B.Sc.May 3 2023

Researchers at Umeå University, Sweden, have found that among the many factors that shape the intestinal microbiota composition, diet has a much stronger impact than defensins, which are intestinal defence molecules produced by the body. Instead, they identified a possible role for these molecules in preventing increased blood glucose levels after consumption of high-caloric “Western-style diet”.

While the effect of defensins in shaping the adult microbiota composition is rather minor when compared to diet, defensins still have a very important role in protecting us against microbial infections; and our research highlights their protective role against the metabolic complications that can arise after the intake of a high-fat and high-sugar Western-style diet.”

Fabiola Puértolas Balint, PhD Student at the Department of Molecular Biology at Umeå University

She is working in Björn Schröder’s research group, which is also affiliated to Umeå Centre of Microbial Research, UCMR, and The Laboratory for Molecular Infection Medicine Sweden, MIMS, at Umeå University.

The gut microbiota refers to the community of trillions of microorganisms that live inside everyone’s gut. Over the past decades, the abundance of specific bacteria in this community has been extensively studied due to its connection to many diseases, including inflammatory bowel diseases, obesity and diabetes, and even psychological disorders. The microbial community is seeded during birth, after which several internal and external factors help shaping the community to its final composition. These factors include, among others, diet (especially fibre), genetics, medication, exercise, and defence molecules, the so-called antimicrobial peptides.

if (g_displayableSlots.mobileMiddleMrec) {
pushDisplayAd(function() { googletag.display(‘div-gpt-mobile-middle-mrec’); });
}

Antimicrobial peptides can be regarded as the body´s own naturally produced antibiotic molecules. In particular, the largest group of antimicrobial peptides – the defensins – is produced by all body surfaces, including the skin, the lungs and the gastrointestinal tract. Defensins are considered the immune system´s first line of defence against infections but at the same time they have also been thought to be essential in shaping the microbiota composition in the small intestine. However, it was so far unclear how big their effect was as compared to diet, which is known to have a major impact.

To investigate this, the researchers from Björn Schröder lab used normal healthy mice and compared their microbiota composition in the small intestine to mice that could not produce functional defensins in the gut, and then both mouse groups were fed either a healthy diet or a low-fibre Western-style diet.
“When we analysed the microbiota composition inside the gut and at the gut wall of two different regions in the small intestine, we were surprised – and slightly disappointed – that defensins had only a very minor effect on shaping the overall microbiota composition,” says Björn Schröder.
However, the intestinal defensins still had some effect directly at the gut wall, where the defensins are produced and secreted. Here, a few distinct bacteria seemed to be affected by the presence of defensins, among them Dubosiella and Bifidobacteria, likely due to selective antimicrobial activity of the defensins.

“To our surprise, we also found that the combination of eating a Western-style diet and lacking functional defensins led to increased fasting blood glucose values, which indicated that defensins may help to protect against metabolic disorders when eating an unhealthy diet,” says Björn Schröder.
The results suggest that strategies that aim to positively modulate the microbiota composition should rather focus on diet, as modulation of the composition via increased production of own host defense molecules, such as defensins, may have only a small impact on the overall composition. However, it is possible that especially early in life, when the microbiota community is not fully matured yet, defensins may have a stronger effect on the microbial composition. Still, increasing the production of defensins may be a valuable option to prevent the development of metabolic disorders.

The results have been published in the scientific journal Microbiology Spectrum.

Source:

Umeå University

Journal reference:

Puértolas-Balint, F., & Schroeder, B. O. (2023). Intestinal α-Defensins Play a Minor Role in Modulating the Small Intestinal Microbiota Composition as Compared to Diet. Microbiology Spectrum. doi.org/10.1128/spectrum.00567-23.

Microbiology

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

Leave a comment

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

Combining host gene expression profiling and metagenomic pathogen detection from plasma nucleic acid enables accurate sepsis diagnosis

Leave a comment

Bhavana KunkalikarBy Bhavana KunkalikarOct 24 2022Reviewed by Danielle Ellis, B.Sc.

In a recent study published in Nature Microbiology, researchers developed integrated host-microbe plasma metagenomics to facilitate sepsis diagnosis.

Study: Integrated host-microbe plasma metagenomics for sepsis diagnosis in a prospective cohort of critically ill adults. Image Credit: Kateryna Kon/Shutterstock
Study: Integrated host-microbe plasma metagenomics for sepsis diagnosis in a prospective cohort of critically ill adults. Image Credit: Kateryna Kon/Shutterstock

Background

Sepsis accounts for 20% of all fatalities worldwide and 20% to 50% of hospital deaths in the United States. For timely and effective antibiotic therapy crucial for sepsis survival, initial detection and identification of microbial infections are required. However, no etiologic pathogens are identified in more than 30% of cases. Distinguishing sepsis from non-infectious systemic disorders is essential since they frequently appear clinically similar during hospitalization.

About the study

In the present study, researchers created a sepsis diagnostic tool that combined host transcriptional profiling along with broad-range pathogen identification.

At two tertiary care hospitals, the team conducted a prospective observational examination of critically ill adult patients admitted to the intensive care unit (ICU) from the emergency department (ED). Patients were divided into five subgroups based on the presence or absence of sepsis. These patients included those who had: (1) clinically adjudicated sepsis as well as confirmed bacterial bloodstream infection (SepsisBSI); (2) clinically adjudicated sepsis as well as a confirmed non-bloodstream infection (Sepsisnon-BSI); (3) suspected sepsis characterized with negative clinical microbiological testing (Sepsissuspected); (4) patients having no evidence of sepsis and an explanation for their critical disease (No-sepsis); or (5) patients with an indeterminate status (Indeterm).

By conducting ribonucleic acid (RNA) sequencing on whole blood samples, the team first examined transcriptional variations between patients having clinically and microbiologically proven sepsis and those without symptoms of infection. A technique called gene set enrichment analysis (GSEA) detects clusters of genes within a dataset with related biological functions.

A differential gene expression (DE) study across the SepsisBSI and Sepsisnon-BSI groups was conducted to identify further variations between sepsis patients with infections in the bloodstream versus peripheral sites. The team developed a universal sepsis diagnostic classifier based on whole-blood gene expression patterns in response to the practical requirement to diagnose sepsis in SepsisBSI as well as Sepsisnon-BSI patients. The team utilized a bagged support vector machine (bSVM) learning strategy to choose the genes that most successfully differentiated patients with sepsis (SepsisBSI and Sepsisnon-BSI) and those without sepsis (No-sepsis).

A median of 2.3 × 107 reads was acquired after sequencing the RNA from obtained patients whose plasma specimens were available. Furthermore, DE analysis was performed to determine if a biologically plausible signal could be used to differentiate patients who did and did not have sepsis.

Results

Heart failure exacerbation, overdose/poisoning, cardiac arrest, and pulmonary embolism were the most frequently diagnosed conditions in the No-sepsis group. Irrespective of the subgroup, most patients required vasopressor support and mechanical ventilation. Patients in the SepsisBSI and Sepsisnon-BSI who had proven sepsis did not show any difference from No-sepsis patients with respect to age, sex, race, ethnicity, APACHE III score, immunocompromise, intubation status,  maximal white blood cell count, or 28-day mortality. In the group of patients without sepsis, all but one patient demonstrated two or more systemic inflammatory response syndrome (SIRS) criteria.

The study also revealed the downregulation of pathways linked to ribosomal RNA processing and translation along with the upregulation of genes involved in innate immune signaling and neutrophil degranulation in sepsis patients. Using DE analysis, the team found 5,227 genes. The Sepsisnon-BSI cohort displayed enrichment in genes associated with defensins, antimicrobial peptides, and G alpha signaling as well as other pathways. On the other hand, the SepsisBSI cohort showed enrichment in genes associated with immunoregulatory interactions between non-lymphoid and lymphoid cells and CD28 signaling, among other functions.

The bSVM model displayed a mean cross-validation area under the receiver operating characteristic (ROC) curve (AUC) of 0.81. Samples with transcript counts lower than the quality control (QC) threshold had a lower mean input mass than samples with sufficient counts.

Interestingly, a number of differentially expressed genes have been identified as sepsis biomarkers, including increased CD177, repressed human leukocyte antigen – DR isotype (HLA-DRA), indicating a biologically significant transcriptome signature from plasma RNA. In the Sepsisnon-BSI group, plasma deoxyribonucleic acid (DNA) metagenomic next-generation sequencing (mNGS) revealed three out of eight bacterial urinary tract infection (UTI) pathogens and two out of 25 bacterial lower respiratory tract infection (LRTI) pathogens. None of the three patients with severe colitis caused by C. difficile had this pathogen. In eight out of 73 patients with proven sepsis, additional potential bacterial pathogens not identified by culture were found.

Conclusion

Overall, the study findings showed that reliable sepsis diagnosis is facilitated by the combination of host gene expression profiling with metagenomic pathogen identification from plasma nucleic acid. Future research is required to verify and gauge the therapeutic utility of this culture-independent diagnostic strategy.

Journal reference: