Plant Viruses: The Unsung Heroes That Deliver Drugs and Destroy Tumors

Before picking up a pipette, Nicole Steinmetz, today a biomedical engineer at the University of California San Diego (UCSD), picked up a pair of skates gifted by her grandmother. At the age of five, she was already gliding across public parks and the outdoor skating rinks, alternating between roller skating in the summer and ice skating in the winter. Her affinity for skating caught the attention of coaches, and in her early teens, she was drafted for the German National Team in Artistic Roller Figure Skating.

But skating was far from her only love. She aspired to be a scientist. While she was a biology student at RWTH Aachen University in the early 2000s, she sought a laboratory to join for her one-year diploma thesis. One topic caught her attention: using plant viruses to produce pharmaceuticals. “[It] just seemed like the perfect match for my interests,” she said.

I think there are a lot of parallels with being an athlete in general and being a scientist.

—Nicole Steinmetz, University of California San Diego

She approached the speaker, molecular biotechnologist Ulrich Commandeur, and swiftly joined his research group for her master’s degree. The encounter became a defining moment that set the direction of her career.

“I really look at [plant viruses] more as a platform technology that we can engineer and repurpose for various applications. So, I made it my thing to just work with plant viruses,” said Steinmetz.

Harnessing Tiny Plant Viruses for Big Breakthroughs in Drug Delivery and Cancer

Steinmetz hung up her skates in her early 20s to fully immerse herself in the wonderful world of plant viruses. “[Skating] has certainly taught me a lot of discipline. I think there are a lot of parallels with being an athlete in general and being a scientist. We’re continuously competing for things, like medals and ranks to grants,” she said. “As a scientist, you also want to be creative, and it’s the same for figure skating. You have to have a creative program in addition to jumping and the technical [aspects], but also, the delivery.”

In 2010, she started her own group at Case Western Reserve University School of Medicine, focused on molecular engineering of bio-inspired nanotechnologies. She decided to explore a few different plant viruses that could be used for drug delivery: tobacco mosaic virus (TMV), potato virus X (PVX), and cowpea mosaic virus (CPMV), which infects black-eyed pea plants.

She leveraged bionanoparticles, also known as viral nanoparticles (VNPs), from these viruses as tools for cargo delivery.¹ A special feature of these plant viruses is that they interact with mammalian cells without infecting or replicating; instead, cells engulf the viruses and recognize them as foreign, which prompts an immune response.

First, Steinmetz investigated how their distinct features, such as differences in morphology, could support drug delivery. For instance, some shapes enable cargo to be loaded either internally or externally while size affects the payload amount.

As TMV is one of the most studied plant viruses, she used it as a reference point for comparison. It has a long, rigid shape. PVX is a less studied subject and boasts a unique shape. Most other particles are spherical or rod-shaped, and often these tend to be short molecules. “But the potato virus acts almost like spaghetti. It’s very long and filamentous,” said Steinmetz.

Steinmetz conducted comparative studies in which she tagged different particles with distinct colored dyes and injected them into an animal to observe the particles’ behavior. The results revealed striking differences. “We very quickly realized that they all behave totally differently,” she explained. “Even viruses that come from the same plant…they have different strengths and weaknesses for different applications.”

Due to their elongated shape, Steinmetz noted that TMV and PVX were better at delivering cargo somewhere within the body. However, CPMV, which is icosahedral shaped, produced an unexpected result. When delivering a drug to cancer cells in mouse models, Steinmetz and her team found the expected empty particles afterward, indicating that the cargo had been successfully released.² The drug-loaded CPMV reduced tumor growth; however, analysis of the tumor growth curves revealed that the empty, drug-free CPMV also exhibited a subtle yet measurable effect. This suggested that the native CPMV influenced the tumor in some manner. Steinmetz explained, “We reasoned that it's probably [got] something to do with the immune system, but we…didn't look at this from the right angle.

Soon after, around 2014, Steinmetz met Steven Fiering, an immunologist and cancer biologist at Dartmouth University, at a conference. Fiering was studying intratumoral immunotherapy, an approach based on the idea that tumors are immunosuppressive and can be manipulated to become immunostimulatory, thereby generating antitumor immune responses—a strategy described as in situ vaccination.

She's remarkable. She's a very exceptional scientist that I've been very fortunate to work with for the past 10 years.

—Steven Fiering, Dartmouth University

Fiering used bacteriophages to treat cancer cells as one approach. He struggled with reducing the amount of lipopolysaccharide (LPS) contamination in his phage preparations. When he attended Steinmetz’s talk on plant viruses, she noted that plant viruses are generally LPS-free because they are derived from plants.

This immediately caught Fiering’s attention. He had never considered using plant viruses for cancer therapy previously, but Steinmetz was enthusiastic about the idea, he recalled. Steinmetz added, “Everything sort of was aligned. We already had some data and some clues.” They planned to study all the plant viruses in Steinmetz’s arsenal, but because Fiering did not have a license from the United States Department of Agriculture to work with infectious plant viruses in his lab, they had to improvise. Steinmetz provided him with versions of CPMV that lacked RNA and were therefore noninfectious to plants. This quick solution also ended up being a serendipitous choice for their project. CPMV produced promising results, while starting with other particles might have discouraged the team from continuing.

Building on Steinmetz’s earlier observations, the duo collaborated to discover that inhalation of CPMV nanoparticles produced anti-tumor effects in a lung melanoma and ovarian cancer mouse model.^3,4 “Sometimes, it's the things that don't work exactly as we think they should be working, those are in fact the most interesting ones,” Steinmetz said. It was pure luck, Fiering said. They found that, compared to other plant viruses, such as TMV and PVX, CPMV was significantly more effective, though the researchers still did not fully understand why.

Fiering remarked, “She recognized plant viruses as their own interesting biological niche,” and she leveraged these malleable viruses for different applications. “She's remarkable. She's a very exceptional scientist that I've been very fortunate to work with for the past 10 years.”

Cowpea Mosaic Virus: An Unexpected Cancer Vaccine Candidate

Although Steinmetz did not have formal immunology training, she and Fiering continued to further investigate plant viruses as an in situ vaccine therapy. CMPV showed promising preliminary results, but the exact mechanism behind its success was still a mystery.

Over the next decade, Steinmetz and her colleagues dug deeper. They found that the CPMV nanoparticles, while not infectious to mammalian cells, can be taken up by immune cells. There, Toll-like receptors recognize them as foreign. So, when injected directly into a tumor, the nanoparticles fire up the immune cells to launch an attack against the cancer cells.⁵

Next, Steinmetz wanted to extensively characterize CPMV with the goal of eventually bringing the platform to the bedside. She collaborated with the Nanotechnology Characterization Laboratory (NCL), whose mission is to support extramural researchers like her.

Marina Dobrovolskaia, the laboratory co-director, director of operations, and head of the immunology section at the NCL, helped work on this project, which began at the peak of the COVID-19 pandemic. But despite being on opposite coasts of the United States, Dobrovolskaia and Steinmetz worked smoothly. “This collaboration is a great example of demonstrating that space doesn’t matter. Science finds a way,” said Dobrovolskaia.

Dobrovolskaia and her team conducted extensive in vitro immunological assays, toxicity studies, and physicochemical characterization of CPMV. She noted Steinmetz’s enthusiasm and commitment to her work throughout the project.

Although the collaboration resulted in several publications, Dobrovolskaia emphasized that the primary objective was to fill critical knowledge gaps and develop characterization tools to help move the research closer to clinical translation. As part of this effort, the NCL hosted one of Steinmetz’s graduate students, Anthony Omole, who was deeply involved in immunological studies examining mechanisms of action, safety, toxicity, and physicochemical properties.

When Omole concluded his training, Steinmetz’s team conducted studies demonstrating that intratumoral administration of CPMV stimulated interferon production, thus activating immune cell functions. Innate immune cells migrated to the tumor microenvironment, leading to tumor cell death. In contrast, the related virus induced pro-inflammatory interleukins but did not produce the same anti-tumor effects.⁶ These findings were consistent with Dobrovolskaia’s team’s findings.

And while much of this work involved mouse models, Steinmetz also found promising results in companion dogs. The researchers injected CPMV into dogs with metastatic melanoma and saw that the treatment relieved immunosuppression and potentiated antitumor immunity.⁷

“This works very similarly to the way good treatments work in humans in that there's variability between dogs,” said Fiering. He noted that the CPMV treatment either shrank or eliminated both treated tumors and distant untreated metastatic tumors, which is the goal of intratumoral immunotherapy.

According to Steinmetz, the CPMV project has been one of her most advanced undertakings.⁸ “The collaboration was very successful, because not only did we answer a lot of research questions and fill these knowledge gaps, but we also answered critical questions which…[brought] this concept [closer] towards clinical trials,” added Dobrovolskaia.

Plant Viruses to Tackle Problems on Earth and in Space

Outside of using plant viruses for cancer immunotherapy, one of Steinmetz’s ongoing projects focuses on the fight against plant-parasitic organisms—particularly nematodes, tiny roundworms that cause an estimated $160 billion in crop damage worldwide.

At an American Chemical Society meeting, Steinmetz attended several lectures about chemical modifications to pesticides to give the drugs better soil motility. “That's sort of where I thought we could maybe use the drug delivery approach, by attaching it to the plant virus,” she said. Plant viruses use nematodes as a vector, transferring between plants by hitching a ride. Although using a plant virus to treat a plant disease may seem counterintuitive, Steinmetz noted, “If the plant virus indeed has good soil mobility, then that might be a good carrier to use…we’re turning the villain into the superhero.”

In recent years, Steinmetz and her team have used protein nanoparticles from tobacco mild green mosaic virus (TMGMV). TMGMV is a plant virus that has already been approved by the Environmental Protection Agency as a bioherbicide, and this virus naturally interacts with nematodes. The researchers used nanoparticles to deliver the drug ivermectin as well as encapsulated double-stranded RNA, which triggers RNA interference and results in potent gene silencing.^9-11 By applying this ivermectin treatment as drip irrigation on the soil, Steinmetz and her colleagues recently completed trials on tomato plants infested with nematodes. This was in collaboration with microbiologist Erin Rosskopf from the Agricultural Research Service. “The results are very promising,” Steinmetz added.

Beyond strategies for using plant viruses in agriculture on Earth, Steinmetz began thinking about how to apply plant viruses or plant molecular farming in space.

The rationale behind that was, “You could manufacture virus-based therapeutics, like vaccines, immunotherapies, and other protein-based drugs in plants in space.” Plants are particularly well suited for this purpose: They are easy to grow, do not require sterile conditions, and are free of human pathogens. She proposed to develop methods that enable researchers to easily manufacture biologics and pharmaceuticals under space conditions.

To do this, Steinmetz uses a random positioning instrument that constantly moves in any direction with varying speeds to simulate microgravity for Earth-bound experiments. "Yeah, it's not truly weightless, but it really reduces the pull of gravity because of the way it's spinning."

Whether applying plant viruses to cancer immunotherapy, agricultural innovation, or the possibility of pharmaceutical manufacturing in space, Steinmetz wears many hats—all driven by an innate curiosity.

“I feel like there's still so much [that’s] unknown,” Steinmetz said. “[The] more we know, the more questions there are, which [makes this] a really exciting time to be in the [field], right? If you answer a question that leads to many more, that's [what makes] being a scientist fantastic,” said Steinmetz.