If the human body is a fortress, mucosal tissues are its rare weak spots. That’s not to say that these humid and sticky surfaces, which include the lining of the mouth, nose, urogenital tract, stomach, and lungs, don’t put up a fight. Mucus-secreting cells keep most invaders out, but every once in a while, some wily microbes breach the mucosal defenses to enter the body and cause illness.
While vaccines can prevent many of these infections, they typically can’t block pathogens from breaching our mucosal barriers in the first place. This is because most vaccines don’t mount an immune response there. These vaccines enter the body via an intramuscular injection, and the vaccine antigens stimulate dendritic cells in the muscle. Those dendritic cells then drain into the lymph nodes to propagate the immune response throughout the body, but that immune protection doesn’t reach the mucosal tissues. Prime examples of this phenomenon are the vaccines developed for COVID-19.
“For COVID, it's transmitted through the upper respiratory tract, so it's really important to establish immunity in the lungs and the nose,” said Brittany Hartwell, an immuno-engineer at the University of Minnesota. “Parenteral vaccines are those that are injected subcutaneously or intramuscularly. They're not really designed to establish immunity in mucosal tissues, so we can think of that as a backup immunity that's designed to protect us against severe disease and death.”
To prevent pathogens from getting into the mucosa, researchers have developed mucosal vaccines. These vaccines enter the body via the same route the pathogens do — directly across the mucosa. Some examples include nasal vaccines, which stimulate an immune response in the nasal mucosa, and inhaled aerosolized vaccines that travel to both the lower and upper respiratory mucosa when a person breathes them in by mouth.
The main challenge with creating effective mucosal vaccines is getting the vaccine across the sticky mucus surface.
“Mucosal surfaces are designed to keep things out efficiently. You're constantly turning over the mucus in your airways and nasal passages very rapidly, and that material gets cleared,” said Darrell Irvine, a bioengineer and immunologist at the Massachusetts Institute of Technology. “If you get through the mucus layer, you still have to get through the epithelial barrier, and that's also designed to not allow things in because you don't want pathogens getting in. So, coming up with ways to make a vaccine that overcomes those barriers has been a challenge.”
A few mucosal vaccines have stood up to the task, including the nasal influenza vaccine FluMist Quadrivalent. But the currently available mucosal vaccines are live-attenuated or inactivated-whole cell vaccines, which can suffer from poor stability and safety concerns (1). People who are immunocompromised, pregnant, or organ transplant recipients, for example, shouldn’t receive these vaccines (2).
“There's really a need to develop something like a subunit protein vaccine that can be taken up mucosally, but the challenge is how can you get them across the mucosal barriers? Historically, that's been the biggest hurdle for development of these types of vaccines,” said Hartwell.
Researchers are now engineering new mucosal vaccines that are just as safe as their intramuscularly-delivered counterparts but that can also mount a strong immune response at the mucosa. With these next generation mucosal vaccines, scientists hope to not only prevent severe disease, but to block infection all together.
From TB to COVID-19
For Zhou Xing, an immunologist at McMaster University, the road to better mucosal vaccines started with tuberculosis (TB). He and his team have been investigating how the body defends itself against a TB infection for more than 15 years. While a TB vaccine called the Bacillus Calmette-Guérin (BCG) vaccine exists, it is formulated as an intradermal injection (3).
“Through our basic research, we learned, actually, that route of immunization is not ideal at all because that route of immunization wouldn't be able to provide us with a very much needed respiratory mucosal immunity,” Xing said.
To target the mucosa, Xing and his team developed an inhaled aerosolized vaccine against TB on an adenovirus vector (AAV). They then compared the immune response generated by the vaccine when they delivered it to people as an aerosolized vaccine by mouth or as an intramuscular injection (4).
“Even with the same vaccine we compared, it's day and night difference in terms of the magnitude of respiratory mucosal immunity,” said Xing.
The inhaled vaccine stimulated both a mucosal and systemic immune response, but the intramuscularly injected vaccine only led to a systemic response. As Xing and his team were finishing up their Phase 1 clinical trial of this vaccine, the COVID-19 pandemic hit. But rather than get discouraged, Xing saw COVID-19 as a crucial opportunity to shift gears and develop a mucosal vaccine for this new virus. Because multiple new SARS-CoV-2 viral variants have emerged since the start of the pandemic, Xing and his team developed what they call a next-generation COVID-19 vaccine strategy (5).
“It's not only from a different route or different delivery method perspective. It's all about the vaccine antigen design,” said Xing. “It expresses three select SARS-CoV-2 antigens including a spike antigen, but more importantly, it includes two additional internal viral antigens.” Because the SARS-CoV-2 virus expresses these two antigens internally, the virus is less likely to mutate in response to the vaccine, and it will remain effective against different SARS-CoV-2 variants. “The idea behind our next generation vaccine strategy is that going forward, we don't have to constantly, regularly update our vaccine antigen design,” Xing added.
He and his colleagues are testing their next-generation inhaled mucosal COVID-19 vaccine in a Phase 1 clinical trial right now, and they hope to have results to share soon. They are currently planning a Phase 2 study for early 2024. While his team’s mucosal vaccine for TB is on the back burner at the moment, Xing hopes to advance that vaccine into additional clinical trials as well. Most of all, he’s excited to develop mucosal vaccines that can stop all sorts of respiratory pathogens in their tracks.
“Hopefully going forward, not only can such a vaccine strategy be applied to controlling COVID-19 but also tuberculosis and other important respiratory pathogens like measles [and] pneumococcus influenza,” he said.
Hitchhiking to halt HIV
Unlike Xing, Irvine’s path to developing mucosal vaccines didn’t start at the mucosa. Instead, his main interest centered on improving the immune response to intramuscular vaccines.
Irvine wanted to send intramuscular vaccines to the lymph nodes more effectively, thereby enhancing the immune response to the vaccine. As he and his team pursued that goal, they discovered that the size of the vaccine carrier dictated whether it would move into the bloodstream or into the lymph nodes. Small peptides trafficked to the blood, but larger molecules went to the lymphatics (6). One of these large molecules is the abundant serum protein albumin.
“Albumin is just the right size that it is constantly coming out of the blood, going into tissue, and then leaving the tissue by going into lymph vessels. So, we realized that albumin itself might be a really interesting chaperone to carry vaccine molecules, and that led to the idea of looking for ways to make a molecule that would interact with albumin,” said Irvine.
Based on this idea, he and his team developed a new vaccine platform that they named an amphiphile vaccine (6). Their vaccine had an antigen attached to an amphiphile tail, which contained both a hydrophilic polymer and a lipid. They found that when they injected this amphiphile vaccine subcutaneously in mice, the lipid part of the amphiphile tail bound albumin. Hitchhiking on the albumin, the vaccine made it into the lymph nodes and enhanced the antigen-specific T cell responses there.
When Hartwell joined Irvine’s team as a postdoctoral researcher with an interest in engineering mucosal immunity, Irvine wondered if they could use albumin to make better mucosal vaccines. To use this albumin hitchhiking approach in the mucosa, Irvine and his team took advantage of the fact that in mucosal tissues, the neonatal FC receptor (FCRN), a molecule broadly expressed on multiple types of mucosal epithelial tissues, binds albumin and shuttles it across the mucosa (7). Once across, albumin then takes the vaccine antigen into the lymphoid tissues to stimulate an immune response. In their case, the researchers delivered the vaccine to the mucosal tissue in the nose so that once it crossed the nasal mucosa, it would travel to the nasal associated lymphoid tissues (NALT) to block infection by pathogens trying to sneak into the body via the nose.
Hitchhiking onto albumin gives this vaccine an additional advantage. “The fact that this albumin binder is itself a lipid actually has another role, which is when it gets into that lymphoid tissue, the lipid tails will insert in cell membranes and paint the immune cells sitting in there. That just helps hold the antigen around longer so that the vaccine response has time to recognize it,” Irvine added.
Hartwell, Irvine, and their colleagues administered this nasal mucosal vaccine against HIV to mice and nonhuman primates, and in mice, they saw strong immune responses in both the mucosa and in the bloodstream (8).
“The HIV work was super exciting because we had never seen that strong of mucosal immune responses in any of our studies before from really — I don't think any of the vaccines that we had tested in lab,” said Hartwell.
Even more astounding, though, was that the strong antibody response in the mouse nasal mucosa triggered an additional antibody response in the mouse vaginal mucosa, a phenomenon called common mucosal immunity.
The HIV work was super exciting because we had never seen that strong of mucosal immune responses in any of our studies before from really — I don't think any of the vaccines that we had tested in lab.
- Brittany Hartwell, University of Minnesota
“There've been a few small trials in humans [showing] that intranasal immunization can elicit antibodies at other mucosal surfaces, but we were pretty struck by how strong and long lived that antibody response was,” said Irvine. “It would be really interesting, for example for HIV, if you could give an intranasal vaccine and get reproductive tract or genital urinary tract antibody.”
As Irvine and Hartwell collected the results of their nasal HIV vaccine, the COVID-19 pandemic struck. As the world shut down, Hartwell pored over the new COVID-19 papers as they came out. She noticed that the COVID-19 vaccine antigens under development looked very similar to the ones she had been using for her mucosal HIV vaccine. She figured, why not see if this same approach could work as a mucosal vaccine for COVID-19? The effort paid off.
Hartwell’s COVID-19 mucosal vaccine led to strong neutralizing antibody responses in both the serum and the respiratory mucosa in mice.
“That 12 months of work spanning from spring of 2020 to spring of 2021 was probably some of the most exciting work of my whole postdoc experience,” said Hartwell.
Hartwell now has her own laboratory at the University of Minnesota where she continues developing intranasal mucosal vaccine platforms, and she wants to use the technology to develop vaccines against many other infections including influenza and cytomegalovirus.
“It's so exciting and really interesting to be able to engineer new things and study the immune response,” Hartwell said. “I can't think of many more fascinating things to study than immunology and how engineering can interface with it.”
- Lycke, N. Recent progress in mucosal vaccine development: potential and limitations. Nat Rev Immunol 12, 592-605 (2012).
- Centers for Disease Control and Prevention, National Center for Immunization and Respiratory Diseases (NCIRD). Live Attenuated Influenza Vaccine [LAIV] (The Nasal Spray Flu Vaccine). (2022). https://www.cdc.gov/flu/prevent/nasalspray.htm
- Okafor, C.N., Rewane, A., and Momodu, I.I. Bacillus Calmette Guerin. StatPearls [Internet] (2023).
- Jeyanathan, M. et al. Aerosol delivery, but not intramuscular injection, of adenovirus-vectored tuberculosis vaccine induces respiratory-mucosal immunity in humans. JCI Insight 7, e155655 (2022).
- Afkhami, S. et al. Respiratory mucosal delivery of next-generation COVID-19 vaccine provides robust protection against both ancestral and variant strains of SARS-CoV-2. Cell 185, 896-915.e19 (2022).
- Liu, H. et al. Structure-based programming of lymph-node targeting in molecular vaccines. Nature 507, 519–522 (2014).
- Rakhra, K. et al. Exploiting albumin as a mucosal vaccine chaperone for robust generation of lung-resident memory T cells. Sci Immunol 6, eabd8003 (2021).
- Hartwell, B.L. et al. Intranasal vaccination with lipid-conjugated immunogens promotes antigen transmucosal uptake to drive mucosal and systemic immunity. Sci Transl Med 14, eabn1413 (2022).