Art Courtesy of Kara Tao.
Traditional vaccines, such as those developed against smallpox and tetanus, have relied upon the introduction of weakened or inactivated pathogens into the body to stimulate the immune system, effectively priming it to recognize and counteract these pathogens in the future. For several decades, however, scientists have pursued an ambitious mission to harness the untapped potential of messenger RNA (mRNA) as a replacement for the pathogens in vaccines. By introducing mRNA, a small piece of genetic material that instructs cells to produce part of a pathogen, the vaccines would theoretically trigger an immune response without causing disease. Scientists envisioned mRNA vaccines harnessing the body’s own cellular machinery to combat pathogens. Their collective efforts, spanning years of research, pushed mRNA vaccine technology to the brink of reality.
Then came the COVID-19 pandemic, a crisis of unprecedented proportions that necessitated a rapid global response. In a mere eleven months, Pfizer/BioNTech produced the first mRNA vaccine to ever achieve full FDA approval for use in the United States. As of September 2023, over eighty percent of the U.S. population has received at least one dose of an mRNA COVID-19 vaccine, fundamentally altering the pandemic’s trajectory and saving millions of lives in the U.S. alone. Yet the quest for innovation continues. In a study recently published in Science Translational Medicine, a team of Yale scientists ventured into a new frontier in vaccinology: the development of a nasally administered COVID-19 mRNA vaccine using nanoparticles.
The Promise and Pitfalls of Respiratory Delivery
Current intramuscular mRNA vaccines, typically injected into the upper arm, excel at activating immune defenses in the bloodstream, but they are not as effective in rallying protective responses in the upper airway and lungs. Thus, for a viral respiratory illness like COVID-19, the allure of an inhalable mucosal vaccine stems from its geographical advantage. When a virus enters the body through the nasal route, the respiratory mucosa (the lining of the respiratory tract) becomes the primary battleground for early encounters. Notably, the Omicron variant has been recorded in higher concentrations in the lungs than in the rest of the body. According to Benjamin Goldman-Israelow, an assistant professor of internal medicine at the Yale School of Medicine and one of the authors of the paper, mucosal vaccines are better designed to engage the immune system precisely at this entry site, enhancing the body’s ability to mount a swift and targeted response there.
The effectiveness of the oral polio vaccine, which played a significant role in the global effort to eradicate polio, is grounded in the same principle. Following ingestion, the vaccine induces a strengthening of immune defenses within the virus’ favored environment—the gastrointestinal tract. This localized approach minimizes the delay associated with the migration of immune defenses from the bloodstream to the environment of interest, thereby reducing the window of vulnerability and bolstering protection against invading pathogens.
While the promise of inhalable vaccines is compelling, it is not without its challenges. Only one mucosal vaccine currently exists to combat pathogens entering through the nasal route: a nasal spray comprising of weakened flu viruses known as FluMist. While this nasal spritz proves reasonably effective in children—occasionally even surpassing the performance of its injected counterpart—its potency wanes significantly in adults. This may be because the pre-existing immunity built up over a lifetime of influenza exposure can inhibit the vaccine’s effects before it can establish new protection, according to Goldman-Israelow. Thus, developing a mucosal vaccine tailored for respiratory viruses presents a unique challenge, and there is no well-established template to follow.
Mark Saltzman, the Goizueta Foundation Professor of Biomedical Engineering at Yale and a senior author of the paper, shared that there were several fundamental challenges in devising an effective mucosal vaccine. The effectiveness of mucosal vaccines relies heavily on how well they can reach and activate immune cells in the mucosal surfaces. To reach cells in the lungs, the vaccine must be able to overcome physical barriers, such as cilia and mucus, meant to prevent debris and pathogens contained in inhaled air from reaching the lungs’ small air sacs, or alveoli. Phagocytic cells, which actively participate in the body’s immune surveillance by destroying microbes and debris, introduce another obstacle. These cells may engulf vaccine particles, thwarting their intended journey to the site of action and potentially compromising the vaccine’s effectiveness.
Finally, respiratory mucosa is especially prone to producing unwanted immune reactions. While current mRNA vaccines employ small fat-based capsules called lipid nanoparticles (LNPs) as their delivery vehicles, these components have been noted to incite inflammation when administered via nasal routes. In the development of nanoparticles tailored for inhalation, the team would have to maximize mRNA delivery efficiency while minimizing detrimental inflammatory responses in the respiratory tract.
Polymers are molecules formed from repeating smaller chemical units known as monomers. Visualize them as molecular chains built from identical building blocks repeated in succession, much like LEGO bricks assembling into a chain. The Saltzman group designs and tests incredibly tiny nanoparticles made from polymers for drug and gene delivery to treat cancers and other diseases. When the COVID-19 pandemic struck in 2019, Saltzman began thinking about how this technology could be applied to inhalable vaccines. He drew inspiration from the work of Akiko Iwasaki, the Sterling Professor of Immunobiology at Yale and a senior author on the study, who is a leading expert on the mucosal immune response.
In 2020, Saltzman’s lab began working on this project and produced biodegradable polymers, called poly(amine-co-ester) (PACE), which can form so-called “polyplexes” with mRNA. The PACE polymers represent a third-generation polymer-based delivery system for nucleic acids like mRNA, distinct from the lipid nanoparticles (LNPs) commonly used in vaccines. The conventional approach in the field has involved employing hydrophobic, or water-resistant, polymers, which have proven somewhat successful in other drugs for delivering nucleic acids. However, these early polymers were prone to becoming positively charged and associating with negatively charged nucleic acids. Administration of these agents could inactivate enzymes, exhibit general toxicity, and affect cell membranes. “The positively charged particles just weren’t well-tolerated in tissues,” Saltzman said.
During the development of the PACE polymers, his team took a different approach. The researchers alternated or substituted some of the positively charged (cationic) groups with hydrophobic groups. This design delicately balanced two forces holding the polymer-nucleic acid complex together: hydrophobic and electrostatic interactions. The hydrophobic component was situated inside the complex, while a mild positive charge resided on the outer surface. The researchers postulated that reducing the charge density within the polymer structure would enhance tolerability and minimize potential side effects. This breakthrough allowed them to create a versatile family of PACE materials compatible with various types of nucleic acids. The researchers found they could fine-tune the polymer’s hydrophobicity and charge based on the specific contents and objectives of their delivery system.
The researchers tested the ability of the PACE-mRNA polyplex delivery system to induce cell-type-specific mRNA expression in the lungs, which would indicate that the system was effective at precisely delivering the nucleic acids to lung cells. Using PACE-mRNA polyplexes in mice, they were able to show that the mRNA was primarily incorporated in epithelial cells lining the airways and antigen-presenting cells in the lungs, which capture, process, and present components of foreign molecules to other immune cells to initiate further responses. The delivery system was successfully used for multiple doses without causing significant inflammation or immune reactions.
To explore the practical applications of the delivery system, the researchers then engineered an inhalable mRNA vaccine encoding the spike protein of SARS-CoV-2, the virus responsible for COVID-19. “With the PACE-delivered mRNA, we were able to see the induction of immune cellular responses within the respiratory tract, as well as in systemic circulation,” Goldman-Israelow said. The intranasal vaccination prompted the production of circulating CD8+ T cells specific to the viral antigen, which serves as a rapid response team, ready to track down and destroy virus-infected cells anywhere in the body.
In the lymph nodes, the vaccine stimulated the formation of germinal centers, which are specialized areas where immune cells undergo intense training and maturation. This training process resulted in the expansion of memory B cells, which “remember” the virus’ unique features, enabling the immune system to recognize and neutralize it more effectively upon future encounters.
The researchers found that the vaccine also led to the production of antibody-secreting cells (ASCs), another critical group of immune cells. ASCs are responsible for manufacturing antibodies, which are proteins that can specifically target and disable the virus. The combined action of memory B cells and ASCs enhances the body’s ability to fend off the virus. Collectively, these findings illustrate the practical applicability of PACE polyplexes for delivering mRNA therapeutics to the lungs.
Since most individuals have already either contracted SARS-CoV-2 or received an mRNA COVID-19 vaccine, the focus is now on providing booster shots that can keep up with new variants. According to Saltzman, this plays to the nasal vaccine’s strengths. “The beauty of the whole thing is that you wouldn’t have to change the delivery system; just exchange the mRNA,” Saltzman said.
Goldman-Israelow, who is also a practicing physician, shared a similar perspective. “Looking more long-term, we know that vaccine hesitancy plays a big role… If we can get intranasal booster-type vaccines going, especially for respiratory illnesses, these will enhance protection and reduce transmission.”