To help meet this challenge, researchers from the School of Science at IUPUI are developing a new biosensor with the potential to achieve the speed and efficiency required for the future of COVID-19 testing.

The work was recently reported in Applied Material Interfaces, a journal of the American Chemical Society. It is led by Rajesh Sardar, a professor of chemistry and chemical biology in the School of Science, and Adrianna Masterson, a graduate student in Sardar’s lab at the time of the study.

“Everyone is chasing high-throughput testing; this type of high-speed analysis is essential to the future of the fight against COVID-19,” Sardar said. “There are many advantages to our technology in particular: It’s fast, efficient, accurate and unprecedentedly sensitive.”

In terms of speed, the COVID-19 test from Sardar’s lab can currently analyze samples from 96 individuals in under three hours, he said. In terms of efficiently, the system requires only 10 microliters of blood.

By comparison, a typical blood panel order by a primary-care physician collects 10 milliliters of blood — more than 1,000 times more.

The sensor also works with other sample types, such as saliva, Sardar said. But the study was conducted using blood since it’s the most complex bodily fluid and therefore the best indicator of a sensor’s accuracy. All test samples were obtained from the Indiana Biobank, which provided 216 blood samples, including 141 samples from patients with COVID-19 and 75 healthy control samples.

Based upon a blind analysis, IUPUI researchers found their biosensor’s accuracy rate was 100 percent and its specificity rate was 90 percent. In other words, the sensor never reported a false negative and only reported a false positive in 1 out of 10 samples. For the purposes of public safety, Sardar said the absence of false negatives is more important than false positives, because a person with a false negative may unknowingly infect others, whereas a person with a false positive is not a danger.

Additionally, Sardar said the sensor was found to be highly accurate at measuring the body’s COVID-19 antibody concentration. This is because it detects not only the virus’s spike protein but also the proteins created by the body to protect against the virus — immunoglobin G, or IgG.

He also said the ability to measure COVID-19 antibodies is significant because many COVI9-19 antibody tests currently approved under the FDA’s emergency use authorization don’t provide specific antibody counts, despite the fact that this number indicates the strength of a person’s immunity to infection.

“Accurately measuring patients’ immunity levels will be critical to protecting against COVID-19 going forward,” Sardar said. “This can be seen clearly in our current state of affairs, as variants like omicron — and, most recently, BA.5 — are infecting even fully vaccinated and boosted individuals.”

To achieve its results, Sardar’s lab’s biosensor uses chemically synthesized gold triangular nanoprisms, which provide a uniquely powerful optical response to even minuscule amounts of IgG. It also means the sensor can detect antibodies in the earliest stages of infection.

The work, which began in the early days of the pandemic, builds upon initial promising results published in June 2021. Next, Sardar aims to further refine the technology, with a goal of eventually being able to process 384 samples in less than an hour — or 5,000 samples per day, if used in a larger testing center.

“This research is about preparing for the future,” said Sardar, who is also a researcher with the Indiana University Melvin and Bren Simon Comprehensive Cancer Center. “The H1N1 strain of the flu is nearly 100 years old. I expect the coronavirus will also be with us a long time. Looking ahead, we need to come up with ways to measure many people’s infections, or risks of infection, quickly, easily and efficiently in order to stay one step ahead of the virus.”

This work was supported in part by the NIH’s National Center for Advancing Translational Science though a grant from the Indiana CTSI.

Protein homeostasis, or proteostasis, is the set of processes that maintain cellular proteins in a functional state, and eliminate damaged proteins that cannot be repaired. Ribosomes are multi-protein molecular machines that synthesize proteins, and mutations in genes that encode ribosomal proteins not only impair protein synthesis but also disrupt proteostasis, leading to chronic proteotoxic stress. That stress, in turn, has a host of cellular consequences and results in delayed development and other irregularities.

To better understand the disorders caused by such disruption of proteostasis, the authors compared gene expression in normal versus ribosomal protein mutant flies during the pupal stage of development. Unexpectedly, they found that a group of genes encoding six gustatory (taste) receptors, called Gr64 genes, were upregulated in the mutant cells.

This finding was unexpected because the Gr64 receptors had previously been known to be present in adult fly neurons, where they help the animal taste sugars, fatty acids, and glycerol. In ribosomal protein mutant flies with just one (rather than two) working copies of the Gr64 genes, there was an increase in cell death via a process called apoptosis. Complete elimination of the Gr64 genes induced multiple morphological defects in the ribosomal mutants, but had little to no effect in cells with normal ribosomal proteins.

Cells bearing ribosomal protein mutants are at a disadvantage compared to normal cells, and are often eliminated in developing tissue where both occur. Mutants carrying only one set of Gr64 genes were even worse off, the authors found, losing out to normal cells at an even greater rate. When the level of Gr64 proteins was variably and quantitatively reduced, the team showed that the most direct effect of reduction was on the function of the proteasome and autophagosome, two different routes by which damaged proteins are removed and recycled.

The connection between promotion of proteostasis and the sense of taste is likely through the molecular mechanism of the Gr64 proteins, which regulate calcium flow; changes in calcium levels are used as a signal transducer in sensory cells, and also regulate multiple proteostatic processes, including both proteosome function and autophagy. Intriguingly, dysregulated and out-of-place gustatory and olfactory receptors have been detected in the affected brain tissue in several human diseases characterized by loss of protein homeostasis, including Alzheimer’s disease and Parkinson’s disease.

“Our work suggests that gustatory receptors play a previously unrecognized role in maintaining protein homeostasis during development in the fly,” Piddini said, “and points more generally toward the connection between calcium-modulating proteins and disorders of protein aggregation and proteotoxic stress.”

Piddini adds, “What I find truly fascinating about our findings is that they underscore the little we understand about how cells adapt to cope with proteotoxic stress. Our experimental strategy could be used further as a way to search and discover additional new proteostasis genes.”

Coauthor Michael Baumgartner concludes, “These findings emphasize an important theme in cell biology: context is everything. It’s easy to see the name ‘gustatory receptor’ and think that’s the end of the story, but evolution can and will repurpose almost anything if it ends up being advantageous. This highlights the possibility that the seemingly hyper-specialized gustatory receptors might be more flexible than previously appreciated.”

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Their findings publish in the July 21, 2022 online issue of PLOS Pathogens.

The research involved building plasmids genetically altered to contain bits of genetic material specifically intended to target a vulnerability in the SARS-CoV-2 virus’s spike protein, a portion of the virus critical to binding and infecting cells. Plasmids are small, circular DNA molecules from bacteria that are physically separate from chromosomal DNA and can replicate independently. They can be used by scientists to transfer genetic material from one cell to another, after which the introduced genetic material can replicate in the receiving cell.

The approach, said senior author Maurizio Zanetti, MD, professor of medicine at UC San Diego School of Medicine and head of the Laboratory of Immunology at UC San Diego Moores Cancer Center, points to the possibility of a more durable, and more broadly effective, COVID-19 vaccine.

“The details are complicated, but the fundamentals are simple,” said Zanetti. “They are based on well-known and proven principles and methods.”

COVID-19 mRNA vaccines, such as those by Pfizer and Moderna, are the result of decades of previous research and development. The pandemic added new urgency, focus and resources. These vaccines promised a faster way to people, though not without significant challenges, such as the need of an ultralow temperature cold chain.

The resulting mRNA vaccines have fundamentally altered the course of the pandemic, dramatically mitigating the severity of disease, hospitalizations and deaths. But notably, said Zanetti, they do little at blocking transmission of the virus. Case rates still rise and fall with the emergence of viral variants.

“The goal at the beginning wasn’t to stop the disease,” said Zanetti. “It was to mitigate the consequences, to reduce COVID’s severity and outcomes. The vaccines have done that. Vaccinated persons tend not to get as sick. They don’t require hospitalization as often. Death rates are down. All of this has greatly reduced pressures on health systems and society, which is a good thing.”

But the ever-evolving nature of the SARS-CoV-2 virus has revealed that the vaccines’ efficacy varies, depending upon variant, often diminishing. The Alpha variant, for example, proved more contagious than the “wild-type” strain that originated in Wuhan, China. The Delta variant was more transmissible than Alpha and Omicron more than Delta. Though the vaccines continue to provide substantial protection against severe disease, the antibodies they induce are consistently less powerful at neutralizing the virus, thus the increased transmission. SARS-CoV-2 continues to be an unrelenting global public health threat.

Zanetti said the newest work emphasizes “quality over quantity,” seeking the induction of antibodies preferentially blocking virus binding to its cell receptor and transmission. This results in a more focused antibody response with the vaccine.

“In the early days of COVID vaccine development, it was about generating a broad, robust immune response,” Zanetti said. “But it was a scattered approach. The vaccines response targeted many epitopes (parts of the virus that the host’s immune system recognizes) and it resulted in an immune response that was largely noise. Most of the resulting antibodies produced didn’t affect the virus’s ability to infect.”

“The new research narrows the focus to a part of the viral spike specifically involved in the virus’s ability to infect that appears to be evolutionarily conserved,” said co-senior author Aaron F. Carlin, MD, PhD, assistant professor in the Division of Infectious Diseases and Global Public Health at UC San Diego Health. In other words, the site doesn’t change with new variants, and represents a persisting site of vulnerability and a reliable vaccine target.

How it works

Zanetti and colleagues built plasmids containing immunogens — molecules that cause B lymphocytes to create antibodies — that were specifically designed to display a knob of the spike protein that is part of the receptor binding motif or RBM. Specifically, these were amino acid residues that act like keys to unlock the cell door. The keys and lock don’t change.

B lymphocytes are part of the immune system. They are prodigious producers of antibodies created to respond and protect against specific antigens or unwanted substances in the body, such as viruses. The average B lymphocyte can spit out 1,000 antibody molecules per second, an incredibly robust production if it is the right antibody for the job.

Zanetti and colleagues cloned the selected spike protein amino acids into a plasmid DNA so that, when injected into the spleen of mice, the introduced immunogen molecules would provoke the production of neutralizing antibodies specifically tuned to the targeted nob on the RBM of the virus protein spike. The researchers then tested their approach on mice with variants of the original SARS-CoV-2 strain (Beta, Delta and Omicron) and found that the immune response was similar across all variants.

“We were a bit lucky in picking our target on the spike,” said Zanetti, “though it was also the result of experience and intuition. I’ve been doing this for 30 years. Earlier experiments by others had suggested this might be a ‘supersite.’ I followed my instincts.”

Zanetti said translating these findings into a vaccine suitable for clinical trials will be “an uphill battle.” There is much invested in current approaches, and it’s a considerable leap from mouse studies to human clinical trials.

But the promise of a consistently effective and easy to administer vaccine is irresistible.

“DNA is very stable. The new ideas for delivery include a pill that survives the digestive system and releases the plasmid DNA to be picked up by B lymphocytes that seem to possess an ancestral property for taking up plasmid DNA. Alternatively, the DNA can be formulated for delivery to the upper airways by suitable formulation for inhalation. Many other researchers and I have investigated and pursued this basic idea before in other ways. It’s time to try it with COVID.”

Co-authors include: Gonzalo Almanza, Alex E. Clark, Valentina Kouznetsova, Andrea Castro, Igor F. Tsigelny, Sandra Leibel, William Bray and Aaron F. Carlin, all at UC San Diego; Eduardo Olmedillas and Erica Ollmann Saphire, La Jolla Institute for Immunology; Yan Wu, Capital Medical University, Beijing, China; and George F. Gao, Chinese Academy of Sciences and Chinese FDA.

A team of researchers including UTHealth Houston faculty members Aaron M. Gusdon, MD, assistant professor in the Vivian L. Smith Department of Neurosurgery with McGovern Medical School at UTHealth Houston; H. Alex Choi, MD, associate professor in the department as well as the Department of Neurology; and Louise D. McCullough, MD, PhD, professor and Roy M. and Phyllis Gough Huffington Distinguished Chair in the Department of Neurology, conducted a multi-site, randomized, double-blind, placebo-controlled, adaptive Phase 2 trial evaluating the safety and efficacy of an anti-inflammatory compound, called OP-101, in patients with severe COVID-19. The results of the trial were published today in Science Translational Medicine.

In the trial, 24 patients classified as having severe COVID-19 across five clinical sites in the U.S. were randomized to receive a single intravenous dose of placebo or OP-101 at 2, 4, or 8 mg/kg. All patients received standard of care, including corticosteroids.

“OP-101 is a novel nanotherapeutic compound that specifically targets activated macrophages and microglia, the primary immune cell in the brain,”said Gusdon, who was first author on the study. “Due to its excellent safety profile, we were excited to offer this therapy to these critically ill patients at Memorial Hermann Hospital.”

Hyperinflammation triggered by SARS-CoV-2 is a major cause of disease severity in COVID-19. OP-101 was found to be better than a placebo at decreasing inflammatory markers, as well as better at reducing markers of neurological injury, including neurofilament light chain and glial fibrillary acidic protein.

Additionally, risk for the composite outcome of mechanical ventilation or death at 30 or 60 days after treatment was 71% for patients receiving the placebo, but just 18% for patients in the pooled OP-101 treatment arms. At 60 days after treatment, 3 of 7 patients given placebo and 14 of 17 patients treated with OP-101 survived.

The data shows that OP-101 was well tolerated in the critically ill patient population and could serve as an effective treatment for patients hospitalized with COVID-19.

“Although this was a small-dose escalation trial, there was clearly a strong signal toward benefit at both acute and chronic timepoint,” Gusdon said. “The possibility that this therapy could also benefit patients with other diseases that lead to systemic inflammatory responses, including various forms of brain injury, is extremely exciting.”

OP-101 is a nanotherapeutic compound that has previously been evaluated in several animal models of inflammatory disease and has shown superior anti-inflammatory and anti-oxidant effects.

The COVID-19 pandemic has resulted in more than 300 million people infected globally, with more than 5 million deaths.

Sujatha Kannan, MD, professor in the Department of Anesthesiology and Critical Care Medicine at Johns Hopkins University School of Medicine in Baltimore, Maryland, was senior author on the study. Other co-authors from Johns Hopkins University included Nauder Faraday, MD, and Rangaramanujam M. Kannan, PhD, both also with the medical school; and Derek K. Ng, PhD, with the Bloomberg School of Public Health.

Also contributing to the study were John S. Aita, MD, with Avera McKennan Hospital in Sioux Falls, South Dakota; Sunil Kumar, MD, with Broward Health Medical Center in Fort Lauderdale, Florida; Ishan Mehta, MD, with Emory University School of Medicine in Atlanta, Georgia; Jeffery L. Cleland, PhD, with Ashvattha Therapeutics, Inc. in San Francisco, California; and Keith Robinson, MD, with Syneos Health in Morrisville, North Carolina.

McCullough is also a faculty member with The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences.