What Bug's in You?

Winter 2021

Say an elementary school has an outbreak of COVID-19, with three confirmed cases in one week. Although quarantining and contact tracing are the best tools we have for ending lines of transmission, those tools can’t always tell you where each of those kids got the virus in the first place. 

And for the school, weighing its options on what to do next, the million-dollar question is: Did these families get the bug on campus or someplace else? 

There’s a good way to answer that, says Vaughn Cooper, Pitt professor of microbiology and molecular genetics. His lab is taking positive COVID-19 tests and sequencing the entire genome of each virus found therein—all 29,000 nucleotides of it. And, using techniques Cooper and his team developed, they’re turning it all around in 72 hours or less. 

Viruses propagate by copying themselves, a less-than-perfect process. In SARS-CoV-2, there’s a new mistake, or mutation, in a given viral lineage about once every two weeks. Because of this, the genomes of the viruses found in cases around the globe have enormous variety—many thousands of distinct flavors. 

When the team sequences a virus, they can compare it “with literally every other sequence ever decoded on the planet,” Cooper said recently on Washington Post Live.  

Take our hypothetical elementary school: If the three virus genomes are identical, “then that’s likely a spreading event, and that might be support for a closure”—to deep clean, test folks en masse and hunt for the culprit within. If the genomes are all different, though, that’s more in line with random infections coming from elsewhere in the community. “You would just isolate those cases and their families, and you could probably keep the facility open,” he says.

Cooper has partnered with the Louisiana Department of Health and Louisiana State University to help quash SARS-CoV-2 outbreaks in that state’s nursing homes, jails and agricultural work sites. And here at home, he’s been using these microbial genome sequencing techniques for years to sleuth out bacterial pathogens within UPMC hospitals in real time. “We were kind of primed to do this when COVID hit,” he says.  

And Cooper’s thoughts on B.1.1.7, the new variant that first turned up in the U.K. in December?

The reason we know about it, he says, is because the U.K. had the foresight to invest in sequencing simply for the purposes of monitoring this pathogen. "This shows how important it is that our country try to rebuild and reinvest in infrastructure like this.
He adds: “All of the primary ways of controlling this virus still matter. And we think that the vaccine is going to work just fine against it." 

Vaughn Cooper talks about predicting evolution and tracking the novel coronavirus in our Pitt Medcast, “Evolving Situation.” 


Image: LSU Health Shreveport Emerging Viral Threat Laboratory and GISAID. Built by Freitas et al with Auspice and Augur. Data made available through GISAID EpiCoV. 

Making Sense of Various COVID-19 Vaccine Technologies


Insight on Inflammation

By Erin Hare

Why do some people with COVID-19 develop severe inflammation? A collaboration between the University of Pittsburgh and Cedars-Sinai has produced a likely answer.
The study, published this fall in the Proceedings of the National Academy of Sciences, uses computational modeling to zero in on a part of the SARS-CoV-2 spike protein that may act as a “superantigen,” kicking the immune system into overdrive as happens in toxic shock syndrome.
Symptoms of a condition in pediatric COVID-19 patients known as multisystem inflammatory syndrome in children (MIS-C) include persistent fever and severe inflammation. While rare, the syndrome can be serious or even fatal. 
The first reports of this condition coming out of Europe caught the attention of study cosenior author Moshe Arditi of Cedars-Sinai, who is an expert on another pediatric inflammatory disease—Kawasaki disease.
Arditi contacted longtime collaborator Ivet Bahar, Distinguished Professor and John K. Vries Professor of Computational and Systems Biology at Pitt, and the two started searching for features of SARS-CoV-2 that might be responsible for MIS-C. Bahar and her team created a computer model of the interaction between the SARS-CoV-2 viral spike protein and the receptors on the foot soldiers of the immune system—T cells. When T cells are activated in abnormally large quantities, as is the case with superantigens, they set off what’s known as a cytokine storm, leading to inflammation.
Using the model, the team was able to see that a region on the spike protein with superantigenic features interacts with T cells. They compared this region to a bacterial protein that causes toxic shock syndrome and found striking similarities in both sequence and structure. “Everything came one after another, each time a huge surprise,” says Bahar. 
“Our research raises the possibility that therapeutic options for toxic shock syndrome . . . may be effective for managing and treating MIS-C in children and hyperinflammation in adult coronavirus patients,” says Arditi. Since the PNAS publication, the Bahar lab has found an antibody specific to the superantigen, which in tests conducted in vitro also interferes with viral entry.  

Wally the llama creates nanobodies that neutralize SARS-CoV-2. (Photo: Sonya Paske at Capralogics, Ltd.)

That’s a Good Llama!  

By Erin Hare and Erica Lloyd

Animals that thrive in environments we think of as extreme have immune systems worth noticing. Think Greenland sharks, quite happy at 7,000 feet below sea level—they live to probably more than 400 years. Think camels who trek for days in the desert. Or their camelid cousins, llamas, guarding livestock and carrying packs in the Andean highlands.
It turns out that llamas create special antibodies, called nanobodies, which are much smaller than human antibodies and—here’s the really good part—many times more effective at neutralizing the SARS-CoV-2 virus. They’re also much more stable. 
“Nature is our best inventor,” says Yi Shi, assistant professor of cell biology at the University of Pittsburgh.
With others here at Pitt and at Hebrew University of Jerusalem, Shi has created a new method to extract tiny but extremely powerful SARS-CoV-2 antibody fragments from llamas, which could be fashioned into inhalable therapeutics with the potential to prevent and treat COVID-19. The breakthrough was reported in the journal Science on Dec 18.
“The technology we developed surveys SARS-CoV-2 neutralizing nanobodies at an unprecedented scale, which allowed us to quickly discover thousands of nanobodies with unrivaled affinity and specificity,” says Shi, senior author.
To generate these nanobodies, Shi and his colleague Yufei Xiang, a research scientist in the Shi lab, turned to a black llama named Wally—who resembles Shi’s black Lab of the same name. The researchers immunized the llama with a piece of the SARS-CoV-2 spike protein and, after about two months, the animal’s immune system produced mature nanobodies against the virus.
Using a mass spectrometry-based technique that Shi has been perfecting for three years, lead author Xiang identified the nanobodies in Wally’s blood that bind to SARS-CoV-2 most strongly. Then, with the help of Pitt’s Center for Vaccine Research, the scientists exposed their nanobodies to live SARS-CoV-2 virus and found that just a fraction of a nanogram could neutralize enough virus to spare a million human cells from being infected.
These nanobodies represent some of the most effective therapeutic antibody candidates for SARS-CoV-2, hundreds to thousands of times more effective than other llama nanobodies researchers have studied for years. Shi’s nanobodies can sit at room temperature for six weeks and tolerate being fashioned into an inhalable mist to deliver antiviral therapy directly into the lungs, where they’re most needed. Since SARS-CoV-2 is a respiratory virus, the nanobodies could find and latch onto it in the respiratory system, before the virus has a chance to do damage.
In contrast, traditional SARS-CoV-2 antibodies require an IV, which dilutes the product throughout the body, necessitating a much larger dose and costing patients and insurers around $100,000 per treatment course.
“Nanobodies could potentially cost much less,” said Shi. “They’re ideal for addressing the urgency and magnitude of the current crisis.”   

(Photo: Aimee Obidzinski/University of Pittsburgh)

A Good Day

Rachel Marini holds up a COVID-19 vaccine vial on Dec. 16, 2020, during the first week of vaccinations in Pittsburgh. Marini is a UPMC clinical infectious disease pharmacist who is on the faculty of the Department of Medicine. She prepared doses of the vaccine so that students from Pitt’s School of Pharmacy could inoculate providers who were slated to vaccinate UPMC employees in the area in the weeks to come. Marini, 31, a native of Bethel Park, says she has been training for this moment her entire career. “This is exactly what I’ve been dreaming of, in all honesty,” she says, tears building in her eyes. “Not to have a pandemic by any means, but to have the ability to help other people.” Marini, who leads the UPMC Presbyterian/Shadyside immunization committee and the Presbyterian pharmacy emergency response team, says the mask she wore hid the smile on her face as she prepared the doses. “It’s a moment that I’m going to keep with me forever,” she says.   —Gavin Jenkins 

Blood Thinners Help Moderately Ill

Many patients who’ve died from COVID-19 formed blood clots throughout their bodies, including in their smallest blood vessels. This unusual clotting causes multiple complications, from lung and other organ damage to heart attacks, pulmonary embolisms and strokes.
A worldwide consortium of clinical trials, coordinated in part by researchers at the University of Pittsburgh, has found that giving full dose anticoagulation treatments, or blood thinners, to moderately ill patients hospitalized for COVID-19 reduced the need for vital organ support—such as the need for mechanical ventilation. The researchers say that adopting the cheap, readily available treatment could help reduce the burden on intensive care units.
Although interim analysis of data from the inpatient trial proved beneficial to moderately ill patients, organizers paused recruitment of critically ill patients in late December due to early signs of futility and a trend toward harm. 

The studies are part of the ACTIV-4 partnership. ACTIV stands for Accelerating COVID-19 Therapeutic Interventions and Vaccines, and ACTIV-4 focuses on the evaluating the role of antithrombotics for treating COVID-19. Incorporated into UPMC and Pitt’s self-learning trial platform, the trials examine progress of those infected with COVID-19 within three groups: outpatients, inpatients and those released from the hospital. Other universities hosting study sites are Harvard University, New York University, the University of Illinois at Chicago and the University of Michigan, among others. 

“This has been an amazingly cooperative endeavor—unequivocally the most rapidly moving, complex but also highly collaborative experience of my life,” Matthew Neal, the Roberta G. Simmons Associate Professor of Surgery, says of the worldwide effort.

Neal is a lead investigator for the inpatient platform. Frank Sciurba, a professor of medicine and education, is a lead on the outpatient platform. Stephen Wisniewski, vice provost for budget and analytics, leads the coordination of the entire ACTIV-4 effort. Maria Mori Brooks, professor of epidemiol­ogy and biostatistics, who codirects the Graduate School of Public Health’s Epidemiology Data Center along with Wisniewski, leads the study design and analysis of the inpatient and outpatient trials.    —Staff Reports 

The new individual biocontainment unit (tested here in a simulation) draws away and traps contaminated air so as not to spread virus. (Photo: Jack Carlson/UPMC)

In the Hood

By Erin Hare

In settings where personal protective equipment (PPE) is in short supply, inserting a breathing tube down a patient’s throat poses a major risk of SARS-CoV-2 exposure for providers as viral particles are released from the airway.
University of Pittsburgh and U.S. Army researchers have created an individual biocontainment unit, or IBU, to keep frontline health care workers safe while they provide care. The device is described in a study published Sept. 3 in the Annals of Emergency Medicine. Earlier attempts to minimize exposure to health care workers involved placing a plexiglass intubation box over a patient’s head and shoulders. With that setup, clinicians place their hands through two large holes in the box to intubate the patient inside. Although such a device may contain the worst of the splatter, it can’t keep aerosols from leaking out. Because of concerns about the potential of airborne viruses to leak from the plexiglass boxes, the Food and Drug Administration has revoked its emergency use authorization for these enclosures.
“Having a form of protection that doesn’t work is more dangerous than not having anything, because it could create a false sense of security,” says David Turer (Res ’20), a plastic surgeon who recently completed his residency at UPMC. Turer was a colead author on the September study.
The Pitt/Army solution, the IBU, is designed to suck contaminated air out of the box with a vacuum and trap infectious particles in a filter before they seep into the room. 
In a simulation, the IBU trapped more than 99.99% of virus-sized aerosols and prevented them from escaping into the environment. 
Several months ago, Turer and colleagues submitted an emergency use authorization application for the IBU; they are preparing to manufacture the devices for distribution as they await approval.
“It intentionally incorporates parts from outside the medical world,” says Turer of the IBU. “So, unlike other forms of PPE, demand is unlikely to outstrip supply during COVID-19 surge periods.” In addition to protecting providers during intubation, the IBU can also provide negative pressure isolation for COVID-19 patients, supplying an alternative to scarce negative pressure hospital isolation rooms, as well as helping to isolate patients on military vessels.
“The ability to isolate COVID-19 patients at the bedside is key to stopping viral spread in medical facilities and on board military ships and aircraft,” said study colead author Cameron Good, a research scientist with the U.S. Army Combat Capabilities Development Command Army Research Laboratory. Devices similar to IBUs were first used by military personnel in the Javits Center field hospital in New York City when New York hospitals were overrun with COVID-19 patients during the first wave of the pandemic. 

Testing the Tests

By Allison Hydzik

There are two main types of tests for SARS-CoV-2, the virus that causes COVID-19: those that tell whether someone has the virus and those that tell whether someone had it.
A recent study by Pitt pathologists sheds new light on the latter type— known as an antibody assay—by comparing the performance of various commercially available tests at different times after illness. COVID-19 antibody assays are mostly used to find out how many people in a given population already had the virus and to determine who can donate convalescent plasma. They also may be important in assessing the effectiveness of vaccines.
“We were surprised to see such large differences in antibody detection both at early and later timepoints,” says senior author Sarah Wheeler, Pitt assistant professor of pathology and associate medical director of clinical immunopathology at UPMC. The study results appeared in the American Journal of Clinical Pathology.
Antibodies are produced by the immune system in response to foreign pathogens. When antibodies against a virus are found in someone’s blood, it’s a telltale sign that person previously encountered the virus.
Wheeler and her colleagues compared six commercial SARS-CoV-2 antibody assays for how well they correctly identify people who are positive or negative for antibodies (otherwise known as a test’s sensitivity and specificity) and the reliability of the test depending on when it was administered after infection.
“We found that some assays detected 20% to 30% fewer convalescent cases than other assays,” says Wheeler. She noted that this could significantly affect studies that seek to determine what proportion of people in a given community have already had the virus.
The team did not specify one test as better than the others, but rather noted that different tests work better in different situations. For example, one test detects antibodies quickly after infection, but does not reliably detect antibodies about a month out, meaning it would be better for determining recent infection compared to infection from an earlier outbreak.