Vaccination has been an undeniable success in reducing the severity and lethality of pathogenic infections. However, the disconnect between the site of vaccination and the route of pathogen exposure, such as the airways in the case of respiratory pathogens, means that many vaccines still struggle to effectively protect against the initial infection. This is evident in established vaccines, such as the acellular pertussis formulation that poorly stopped nasal colonization and the more recent vaccines against COVID-19 in which breakthrough cases, though mild, still occured.^1,2

“What you’d like to have there is a resident memory population that very quickly recognizes that the virus has come in and basically mounts a rapid immune response to eliminate that pathogen before it has a chance to spread to the lungs or to other parts of the body,” said Ulrich von Andrian, an immunologist studying mucosal immunity at Harvard Medical School. Resident memory T and B cells are immune cells that reside in a specific tissue after being activated, providing local protection to that site.^3,4

With rare exceptions like the oral polio vaccine and intranasal vaccines against influenza, the majority of immunizations are administered intramuscularly. However, while intramuscular vaccines produce robust circulating antibody and systemic memory cell responses, these poorly protect the mucosal barriers, such as the respiratory tract. “An improved vaccine would ideally generate a resident memory population in the nasal mucosa or at the site of entry itself,” von Andrian explained.

Because of this discrepancy, many researchers are interested in developing nasal vaccines, and some have shown promise in animal models. One group showed that nasal vaccines induced memory T cells in mouse lungs that were maintained for one year.⁵  Another group observed airway influenza vaccination prompted memory B cells that secrete immunoglobulin A (IgA) into the mucosa; this airway route also provided protection against different strains of the virus.⁶

Despite these benefits, questions about their long-term efficacy and safety have limited human trials. However, research into novel adjuvants and intranasal immunity are helping to close this gap to improve mucosal protection.

Barriers to Intranasal Immunization

One major challenge in targeting the nose and respiratory tract for vaccination is overcoming this region’s many physiological barriers. “Since we’re exposed to so many antigens all day through these surfaces, there’s very unique features of the immune environment of these surfaces,” said Pamela Wong, an immunologist at the University of Michigan.

These features include populations of suppressive T cells and antigen-presenting cells that regulate  local immune responses to minimize activation, creating a tolerant state to avoid overreaction in non-pathological situations.^7-9 This tract also routinely produces mucus to capture and expel much of the content that enters it, while cilia on epithelial cells help to further clear particulates.^10,11 Additionally, the epithelial cells themselves are an obstacle for vaccines. “This barrier tends to be very tight,” von Andrian explained. “Which means that if you just take, let’s say, lipid nanoparticles with mRNA or recombinant protein with an adjuvant, and give that into the nasal mucosa, chances are that you don’t really get an efficient immunization.” For intranasal vaccines to be effective, the antigen must be able to access the immune cells on the other side of the epithelium.

One of the early approaches to overcome these challenges leveraged adenoviruses as vaccine vehicles, since these infiltrants possessed mechanisms to skirt the body’s mucosal defenses.¹² As a bonus, the adenoviruses stimulated the immune system, making them ideal for overcoming these cells’ immune tolerance. These attenuated vectors still posed safety concerns for people with compromised immune systems, though, and early virally-vectored vaccines demonstrated a risk of Bell’s Palsy, a type of facial paralysis.¹³ Additionally, the potential for the immune system mounting memory defenses against them made them less than ideal for repeated applications.^14,15

The Proof is in the Nasal Vaccine Formulation

To overcome these limitations, researchers turned their attention to synthetic vehicles and adjuvants, like oil-in-water nanoemulsions (NEs) coupled with immune agonists.^16-18 However, the formulation of these vaccines is important to ensuring their success. “You have to have the adjuvant in conjunction with the antigen, and they both have to get to the same antigen-presenting cell,” explained von Andrian.

Wong and her colleagues developed one intranasal NE that activates multiple immune receptors.^19,20 “It also carries antigen and helps deliver it across the mucosa better, and it also provides a depot by sticking to the mucus a little bit more,” she explained. This adjuvant was safe and induced antigen-specific antibody responses in the nose and circulation in a Phase I clinical trial.²¹

Recently, Wong and her group collaborated with researchers at the Icahn School of Medicine to combine their NE with an RNA that activates RIG-I, an antiviral immune receptor.²² “By combining our nano emulsion with their RNA agonist, we’ve been able to get a much more tailored immune response to trigger antiviral pathways,” she said.

Using this adjuvant to deliver SARS-CoV-2 spike protein intranasally, the researchers investigated their formulation as a standalone nasal vaccine and as an intranasal booster following intramuscular vaccination with the Pfizer mRNA COVID-19 vaccine.²³ This mimics a “prime and pull” strategy that has been explored in other mucosal models and for COVID-19 booster vaccination.^24,25 The rationale is that the preliminary vaccination induces populations of T cells and B cells that circulate the body looking for an antigen. The secondary vaccination in the respiratory tract calls these immune cells to this area, where the cells take up residence as a memory population to subsequently activate when the antigen enters the nasal or respiratory tract.

While intramuscular prime-and-boost vaccination induced robust circulating antibodies, only immunization strategies that included the intranasal formulation, alone or as a booster, produced mucosal IgA. The team demonstrated the importance of this in their challenge model, where although intramuscular primary and secondary vaccination protected mice from severe disease, only intranasal vaccination, either as a prime and boost or boost after intramuscular prime, prevented viral detection in the upper respiratory tract. Furthermore, intranasal vaccination induced better cross-protection against different SARS-CoV-2 strains.

Mapping Immune Memory to Guide Vaccine Design

While adjuvants can help overcome challenges in inducing respiratory mucosal vaccine responses, researchers are also interested in mechanisms to track the outcomes of a vaccine. “If you vaccinate someone, you need longevity of the vaccine effect,” von Andrian said. “The first challenge is, how do you safely induce a local memory population in the nasal mucosa? And then the second is, how do you maintain that memory population over prolonged periods of time?” Providing answers to these questions can improve vaccine design.

Ziv Shulman, an immunologist at the Weizmann Institute of Science, and his team studied mucosal responses in the gastrointestinal tract using whole-organ imaging. With the COVID-19 vaccine rollout, Shulman said that many studies explored antibody responses and their activity against the viruses, but he had a different question. “Where are the cells? Where are the structures that support the immune response?”  

To apply their skills to a new system, Schulman and his team developed a model of intranasal vaccination to study the development of memory respiratory mucosal cells and identify their niche.²⁶ To isolate vaccine-driven immune responses, they used an ovalbumin immunization model.

Jingjing Liu, a graduate student in Shulman’s group and study coauthor, determined that responding B cells were activated in a compartment of the nasal cavity called the nasal-associated lymphoid tissue (NALT). However, following vaccination, these cells migrated out of the NALT. At first, Shulman and his team struggled to find the final resting place for these cells. “They didn’t go to the lung,” Shulman said. “[Liu] looked everywhere in the mouse, like bone marrow and lymph node, and we couldn’t find the cells.”

Liu wondered whether these cells simply went to a new niche in the nasal cavity. Collaborating with neuroscientists, she mastered the skills needed to fully analyze the cranial bones and found the new location the B cells called home: the bony structures inside the nose called nasal turbinates. “I was shocked,” Liu said. She recalled seeing the population of cells outside of a lymph node, “It’s like fireworks.”

According to Shulman, understanding more about the immune responses during and after vaccination provides researchers with better information to design vaccines. “It gives us another level that we can measure in mice, and also in human,” Shulman said. “Rather than just looking at the molecule at the end point, you get transitional points that you can look at.”

The findings in mice answer questions about where activated immune cells go after stimulation, allowing scientists to better study long-term vaccine responses in novel formulations. Shulman anticipates that there is still more to learn about the molecular mechanisms driving these immune outcomes that can help scientists understand vaccine strengths and weaknesses. “If we can map this information, on a molecular level, we can try to find what kind of vaccine, adjuvant, antigen, [induces these cells], and by this way, solve the problem,” he said.

Next, Shulman and his team want to corroborate their findings in humans. Collaborating with allergy physicians, they hope to collect samples of nasal polyps to try to find similar structures to what they identified in mice.

Studying the Mucosa: Nasal Vaccines and Beyond

Expanding science on mucosal immunity and adjuvants will inevitably help bring more nasal vaccines into clinical trials and hopefully to markets. However, Wong and Shulman both see this knowledge improving mucosal science overall.

“The amount of interest now [in intranasal vaccines] is very exciting, but it’s just the beginning of the potential of what we can do with intranasal and other mucosal delivery systems.” Wong said, highlighting the potential of these platforms to improve therapeutic strategies in mucosal cancers.

Meanwhile, Shulman is working with physicians to extend his team’s findings to develop better treatments against allergic responses, possibly using his group’s findings to help guide interventions. “You can test this drug and see that the cells are disappearing, or you can look at the cells within these bones and see that you get rid of them. And it gives you another measurable parameter to see if your drug or your vaccine or whatever you do is working or not,” he said.