Plants produce ‘green vaccine’ against norovirus in ASU study

Each year, close to 700 million people are stricken with a viral infection that causes vomiting, diarrhea and stomach pain. While the majority will recover in a few days, some 200,000 infected patients will die. The culprit is known as norovirus — often referred to as "the cruise ship illness." Currently, no recommended treatments or vaccines are available.

In a new study, Andrew G. Diamos and Hugh S. Mason of the Biodesign Center for Immunotherapy, Vaccines and Virotherapy describe a trial vaccine against norovirus. The innovative therapeutic is produced using a plant-based system, which offers many advantages over traditional routes of pharmaceutical production.

The study demonstrates that the new plant system for norovirus vaccine production is effective against the tenacious pathogen and that the versatile method could be used for the development of a broad range of novel vaccines. It is estimated that an effective vaccine against gastroenteritis could save billions of dollars in healthcare costs in the U.S. alone.

The researchers outline a technique to produce so-called virus-like particles (VLPs) — portions of the virus able to provoke a strong immune response, without the disease-causing properties of whole norovirus.

"The beauty of VLPs is that they are very simple, but also very effective vaccines. A VLP is comprised of only a single protein, repeated many times: 180 identical copies of this protein self-assemble inside the plants to make the entire structure of the virus,” Diamos said. “Because this structure looks just like the real virus to our immune systems, it makes an excellent vaccine. However, since the VLP is just an empty shell without any viral genes inside, it has no potential to cause infection. Coupled with the inherent safety of plants, this makes plant-made VLP vaccines among the safest known vaccines."

The new study describes the production of virus-like particles derived from norovirus at more than three times the level previously reported in plant-based systems, allowing the production of milligrams of pure, fully-assembled norovirus particles from a single tobacco leaf. Efforts to optimize the VLP expression system also significantly reduced cell death in treated plant leaves.

The group’s findings recently appeared in the journal Protein Expression and Purification.

Insidious invader

Norovirus is the most common cause of gastroenteritis. The highly contagious pathogen, commonly spread during winter months, is usually transmitted through contaminated food or water or through person-to-person contact. Symptoms, including severe diarrhea, stomach pain and vomiting, typically occur within 12 to 48 hours of infection. Dehydration is common.

The aggressive virus is sometimes referred to as “the perfect human pathogen.” It remains highly stable in the environment, can cause infection at very low doses and is shed in large quantity by those infected. Norovirus induces very limited immunity after natural infection, as the virus is rapidly evolving.

Norovirus spreads readily in densely populated, contained areas like ships, though it is a ubiquitous scourge in both developed and developing countries. Norovirus is known to spread through facilities like hospitals, schools and military bases with ferocious speed — often requiring lengthy and challenging decontamination measures.

As the authors note, plant-based vaccines offer a safe, convenient and cost-effective means of producing novel vaccines for a range of biological threats, including norovirus. Such vaccines have advantages over traditionally produced vaccines produced in mammalian or insect cell systems, in terms of cost, efficiency and safety.

Plants to the rescue

In earlier years, plant expression systems for vaccine development were overwhelmingly based on transgenic plants, with permanently altered or recombinant DNA residing in the cell nucleus or chloroplasts. However, transient plant alterations based on infection with viral vectors have now become the norm and have improved convenience and speed, allowing researchers to produce large quantities of protein used for the vaccine in just days. Protein expression in transgenic plants typically requires months.

The most commonly used viral vector has been the tobacco mosaic virus, though the authors demonstrate substantially better results with their system based on the bean yellow dwarf virus as a vector, which is less toxic and damaging to infected leaves.

The vector-based technique has been used to produce vaccines against swine flu, bird flu and many other leading infectious diseases. The vaccines can typically be produced in weeks rather than months, a critical advantage in the case of sudden disease outbreaks, where time is of the essence.

Building a vector

The strategy for producing virus-like particles for norovirus vaccine occurs in several stages. The re-engineered bean yellow dwarf virus contains genes for producing the norovirus VP1 capsid protein in tobacco leaves. This vector is introduced into a bacterium known as agrobacterium tumefaciens.

Leaves of the target tobacco plant are then infiltrated with a solution contacting the bean yellow dwarf virus-bearing bacteria.  When tobacco leaves are exposed to this viral vector system through the process of agroinfiltration, they respond by producing norovirus-derived virus-like particles, which self-assemble in the tobacco leaves. Essentially, tobacco leaves infected with the bean yellow dwarf virus are induced to act as a production factory for the virus-like particles used in the vaccine.

The virus-like particles produced have structural and antigenic characteristics that are recognized by the body as though they are complete norovirus virions, though disease-causing viral components are absent. The resulting vaccine is effective at stimulating both humoral and cellular immunity and priming the body against a subsequent infection by norovirus.

Versatile vaccines

Tobacco plants are hearty and reproduce rapidly, allowing vaccine production to be scaled up according to need, and plant-made vaccine candidates do not require the advanced sterilization and purification steps typical for mammalian and insect cell culture vaccines. Further, producing virus-like particles rather than whole viruses avoids the use of infectious agents in the production process, improving safety.

One challenge facing researchers producing plant-made vaccines is the damage caused to tobacco leaves by the virus used to infect them. The new technique incorporates a number of design improvements in the bean yellow dwarf virus vector, reducing cell death and boosting virus-like particle yield by 65 percent.

With careful optimization of extraction conditions, the group produced norovirus virus-like particles with 90 percent purity, with no losses in yield. The method enables the production of milligram quantities of virus-like particles from a single plant leaf, outpacing earlier efforts by two to three times. The use of bean yellow dwarf virus for the expression system is also desirable as this plant virus has a broad host range and can be used for protein expression in a large number of plant species.