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New research from the University of Cincinnati demonstrates how specially engineered bacteria taken orally can operate as a delivery system for antiviral therapies and vaccines. The research, led by Nalinikanth Kotagiri, Ph.D., is in the journal Gut Microbes.

Kotagiri's lab focuses on engineering probiotic bacteria to accomplish a wide variety of functions, from breaking down cancer's defenses to imaging and diagnosing lung infections.

A few years ago, the team asked whether the same chassis, using the bacterium E.coli Nissle 1917, could ferry antiviral therapeutic agents or vaccine antigens directly to the gut, a major portal of viral entry. The team focused on the COVID-19 virus, SARS-CoV-2, for the proof-of-concept research.

"Oral delivery lets us target the mucosal surfaces where pathogens first gain a foothold while avoiding needles and cold-chain logistics," said Kotagiri, an associate professor in UC's James L. Winkle College of Pharmacy.

Vaccine platform

Most engineered bacteria keep their therapeutic cargo inside the cell, but vaccines work best when antigens are presented to the immune system. The UC team therefore displayed viral proteins on the bacterial surface and harnessed outer-membrane vesicles (OMVs)—nano-sized spheres that bacteria naturally shed—to act as self-propelled delivery vehicles. Once released, OMVs traffic through the gut epithelium, enter blood circulation and distribute their payload to distant tissues.

Nitin S. Kamble, Ph.D., a research scientist in Kotagiri's lab, systematically screened anchor motifs and expression cassettes to optimize antigen density on the probiotic surface. For the vaccine version, the bacteria was designed to express the spike protein found on the surface of the virus that causes COVID-19. This same spike protein is currently delivered through mRNA COVID-19 vaccines.

Kotagiri said current vaccines are safe and effective at providing what is called systemic immunity, as antibodies move throughout the whole body in the bloodstream. But there are gateways in the body where viruses typically enter—through mucosal lining in the gastrointestinal system, lungs and other organs—that can be targeted to provide what is called mucosal immunity.

In preclinical animal studies, a two-dose oral regimen generated blood-borne (systemic) antibody levels comparable to intramuscular mRNA vaccination. Notably, it produced markedly higher levels of secretory immunoglobulin A (IgA) in the gut and airways—the antibodies that underlie mucosal immunity, considered critical for blocking infection at the point of entry.

Therapy platform

While vaccines are delivered before a person is infected with a virus, antiviral therapies such as monoclonal antibodies are given as a treatment after infection.

The team developed another version of engineered E.coli Nissle 1917 to display therapeutic proteins on the surface. To create a post-exposure therapy, the team encoded anti-spike nanobodies: antibodies that are one-tenth the size of conventional monoclonal antibodies.

Although full viral-challenge studies are pending, nanobodies released from the engineered bacteria reached the bloodstream, likely facilitated by OMVs, and accumulated in lung tissue, where they neutralized SARS-CoV-2 in ex-vivo assays.

'"A unique aspect of this approach is the use of OMVs as natural postmasters, efficiently packaging and delivering these therapeutic molecules to their intended targets,"' said Kamble. "OMVs can fuse with host cells and deliver a concentrated payload of therapeutic proteins, making them ideal for mucosal delivery."

Current IV infusions typically deliver a larger quantity of monoclonal antibodies, but because the probiotic can reside in the gut for days or weeks, it offers a self-renewing and sustained depot of antiviral molecules.

Next steps

Now that the team has optimized the bacteria for this purpose, Kotagiri has identified an opportunity for quick adaptation of this platform to develop oral vaccines and therapies for common viruses like influenza and norovirus.

"All that optimization was necessary for us to prove that this is a platform that we can take forward," he said. "If you have a nanobody or antigens against a virus, we can plug that into our construct."

Clinical trials will validate the safety and efficacy of this delivery system for new engineered bacteria targeting other viruses. But Kotagiri said that so far the engineered bacteria have been found to be safe to use and do not generate any adverse immune response or side effects in animal models. Moreover, the parent strain of bacteria has decades of safe use as a probiotic.

"In the future, maybe we can integrate both agents so the same bacteria has both the vaccine and the nanobody therapy components," he said. "But the common denominator is the bacteria, where it has the versatility to do both vaccination and therapy, orally."