When SARS-CoV-2 first began spreading across the globe, not every lab was equipped to study it directly. The virus behind the current pandemic is highly pathogenic and transmissible, leading the US Centers for Disease Control and Prevention to require many of the same biosafety guidelines that shape the study of diseases such as tuberculosis and Ebola.
As in many moments throughout the last year, the scientific community responded by creatively adapting existing tools to the study of COVID-19. Among these, researchers turned to models of the pathogen such as pseudoviruses and chimeric viruses that can be studied safely in labs with lower biosafety level (BSL) clearance than required for studying the wildtype version, in an effort to expand the study of the novel coronavirus. Pseudoviruses don’t replicate, rendering them harmless, but by replacing their surface envelope proteins with those of SARS-CoV-2, researchers can glean insights into the ways the pathogen infects cells. A chimeric virus is made by inserting the genetic material of one virus into the genome of another, safe surrogate, and these introduced sequences are passed on when the virus replicates.
In addition to their safety, pseudoviruses are “extremely versatile in that you can . . . introduce different envelope proteins and you can introduce mutations, which is making it extremely useful for us to screen a lot of different variants,” says Carol Weiss, a virologist who heads the laboratory of immunoregulation at the US Food and Drug Administration. “If you want to introduce mutations in real viruses, it’s a whole lot more work.”
An approximation of the real thing
Pseudoviruses were first developed in the 1960s, after scientists began studying a vesicular stomatitis virus (VSV) isolated from cattle. In addition to replicating well in culture, they later learned that its surface protein, VSV-G, facilitates entry into all eukaryotic cells, making the virus a useful vector not only as a pseudovirus but as a ferry to deliver DNA into cells for therapeutic purposes. The first Ebola vaccine was developed using a VSV platform, and more recently, the virus has been engineered to seek out and destroy cancer cells.
HIV-based platforms, which came about in the 1980s, have since replaced VSV as the most common model for developing both pseudo- and chimeric viruses. Unlike VSV’s negative-strand RNA genome that must be transcribed once inside the cell, HIV’s positive-strand RNA genome can instantly begin translation, making pseudoviruses based on HIV faster to produce. HIV-based model viruses have now been used in many of the same applications as VSV, with scientists applying them to the study of diseases such as AIDS, SARS, MERS, and influenza.
We wanted to really validate that the tool that we generated did appear exactly, with everything we could throw at it, the same way as SARS-CoV-2.—Sean Whelan, Washington University
To harness these surrogates to study SARS-CoV-2, researchers first needed to prove that their pseudo- and chimeric viruses are viable stand-ins for the real thing. SARS-CoV-2 is a uniquely bulky virus—its genome is roughly 30 kilobases, while HIV and VSV sit around 10 kilobases—and while it is more similar to HIV, none of the three are closely related. Fortunately, both HIV and VSV appear to be compatible for making coronavirus models.
Sean Whelan, a virologist at Washington University in St. Louis, is one of many scientists who has developed a viable chimeric virus platform and quantified its performance in the face of antibodies against the real thing. To do this, he developed two complimentary assays—one for use in infectious disease laboratories with the BSL-3 clearance required to handle live SARS-CoV-2 and another for labs working under a lower, BSL-2 clearance—and studied how each virus responded to a battery of different treatments. It wasn’t enough, he says, to test the viruses’ ability to evade just one type of antibody, so he used monoclonal and polyclonal antibodies and serum from recovered COVID-19 patients—as well as a type of ACE2 decoy protein suggested as a possible therapeutic to draw the virus away from the cells’ own receptor. “We wanted to really validate that the tool that we generated did appear exactly, with everything we could throw at it, the same way as SARS-CoV-2.”
The BSL-2 assay Whelan designed uses a chimeric VSV that includes the SARS-CoV-2 spike protein and produces a green fluorescent protein as a signal for infection (luciferase is also often used). Whelan exposed human cells to his chimeric virus until 100 cells had been infected. Then, he bathed the cells in each type of antibody and the decoy protein and recorded how quickly and to what extent the chimeric virus was able to escape them.
Simultaneously, Whelan’s colleague Mike Diamond, an immunologist at Washington University School of Medicine, developed a BSL-3 assay using a live clinical isolate of SARS-CoV-2. Similar to Whelan’s assay, he exposed 100 infected cells to the same litany of tests and used immunostaining to track infected cells over time.
The results, published in Cell Host and Microbe in September, demonstrate that the two assays produce neutralization profiles for each exposure that align 93 percent of the time, meaning that the chimeric virus is a suitable proxy for assessing the ability of antibodies to shut down SARS-CoV-2. Many papers have published similar results, comparing their assays against wildtype coronavirus. This validation against the true virus is important, Whelan says, because a chimeric virus “looks like COVID from the outside, but it’s not COVID on the inside.”
A speedy means for tracking emerging variants
While studies involving true isolates of SARS-CoV-2 will always be the gold standard for understanding the complex dynamics of COVID-19, there are instances in which pseudo- and chimeric viruses are better choices. For one, such models are inherently more genetically stable than their wildtype counterparts. Viruses change a bit each time they interact with a new host, and individual copies within a single person might not be identical. In the lab, viruses can also evolve new mutations in response to the types of cells used to culture them. Viruses propagated in monkey cells, for example, may adopt different mutations than those cultured in human cells.
One of the key advantages is just the ease of use in the laboratory.—Carol Weiss, US Food and Drug Administration
When scientists engineer proxies of SARS-CoV-2, they can control for some of that variation by using plasmids that all contain a single clone of the spike protein. “One of the key advantages is just the ease of use in the laboratory,” the FDA’s Weiss tells The Scientist. “It’s not a mixture of mutations. We know exactly what we’re testing.”
In some instances, researchers specifically want to study new mutations to, say, see how they might change the efficacy of vaccines. Already, notable variants have emerged in the UK, South Africa, and Brazil that dampen the effectiveness of some vaccines. South Africa recently halted the rollout of AstraZeneca’s vaccine after it produced only a weak response against mild and moderate illness, and Moderna announced plans to modify the second iteration of its vaccine in an effort to remain robust against these new variants. For studying such mutations in the spike protein, pseudoviruses offer a much faster model for generating and testing new versions than the actual virus does.
Rather than having to clone each new variant as it emerges in wildtype specimens to assess whether a vaccinated individual’s antibodies will be effective, pseudoviruses allow scientists to quickly create new models with the same mutations to run their experiments. Theodora Hatziioannou, a virologist at the Rockefeller University, says that she routinely screens as many as 1,000 pseudovirus samples within 48 hours to determine whether any are a cause for concern, a process that would take much longer were she waiting for live clinical isolates of SARS-CoV-2 bearing the same mutations. With pseudoviruses, “you are able to test a lot more samples much faster with the appropriate replicates.”
Hatziioannou’s team recently tested the blood of 20 patients who had received either the Moderna or Pfizer vaccine and identified individuals who mounted a robust antibody response to the virus. They extracted and isolated 18 unique antibodies associated with the spike protein’s receptor-binding domain (RBD) and exposed a VSV-based chimeric virus bearing the SARS-CoV-2 spike to each antibody type individually to see which mutations are selects for as the virus replicates.
Next, they engineered HIV-based pseudoviruses with each unique spike mutation that resulted from the VSV experiments and used their own neutralization assay to determine whether the mutations conferred resistance to SARS-CoV-2 antibodies. To confirm that the virus would behave the same when exposed to a cocktail of many different antibodies, as in a human body, the researchers also pitted the pseudoviruses against plasma taken from recovered patients.
Their results, shared on the preprint server bioRxiv on January 19 and recently accepted in Nature, demonstrate that many mutations do decrease the ability of neutralizing antibodies to effectively combat the virus. “In all cases, the mutations identified by the VSV [chimeric virus] were mutations that conferred resistance in the HIV pseudotype,” Hatziioannou says, although she adds that vaccines are still an important tool. When they looked specifically at mutations found in the B.1.351 and B.1.1.7 variants, first identified in South Africa and the UK, respectively, they found that the neutralizing ability “was a bit weaker than a virus without these substitutions, but the effects were overall small.”
Where pseudo- and chimeric viruses fall short
While these surrogate platforms have been useful tools for studying how SARS-CoV-2 enters our cells, there will always be a need for BSL-3 labs that can study the virus directly. “The disadvantage of using pseudotypes is that the only biological property that you’re really going to learn about coronaviruses is those properties that relate to the spike protein,” Vicente Planelles, a pathologist at the University of Utah, tells The Scientist. “As soon as we go beyond the processes that the spike is responsible for, we need a different model.”
No pseudovirus platform can fully recapitulate how the virus propagates and releases new copies of itself, for example, nor can scientists use them to study other mechanisms by which the virus circumvents the body’s immune system.
In addition, while pseudoviruses are useful for assessing the efficacy of vaccines and antibody therapies, they are less helpful with respect to evaluating other drugs that treat the severity of the disease. Remdesivir, for example, was highlighted as a promising option early in the pandemic before conflicting evidence cast its effectiveness into doubt. But the family of drugs to which remdesivir belongs, called nucleoside analogs, could prove useful in preventing extreme illness if scientists could find a better candidate. These drugs work by interfering with a virus’s ability to replicate, which pseudoviruses do not do. “If we found nucleoside analogs that work better than remdesivir, we would be in great shape,” Planelles says. “But you cannot probe that with a pseudovirus, because the gene [that the drug targets] is an internal gene that is not the spike.”