Mast Cell Activators as Adjuvants for Intranasal Mucosal Vaccines

Abstract

Mast cells have roles in immune regulation, allergy, and host response to pathogens. Compounds that activate mast cells (MCAs) can serve as vaccine adjuvants, potentially outperforming current FDA-approved options, especially for mucosal vaccines. While most vaccines are administered intramuscularly, intranasal and needle-free formulations offer benefits like improved compliance and accessibility. However, the lack of effective adjuvants limits mucosal vaccine development. This review explores MCAs as promising alternatives to traditional adjuvants, aiming to enhance mucosal vaccine efficacy. We summarize the nascent work of formulating MCAs like compound 48/80 into nanoparticles, with excipients such as chitosan and chitosan/alginate. Other MCAs like the peptide mastoparan 7 complexed with CpG have formed nanoparticle complexes that illustrate protective mucosal immunity in a model of influenza. The small molecule MCA ST101036, when encapsulated in acetalated dextran particles, has demonstrated enhanced immune responses and protection in a West Nile Virus model of infection. This review highlights the potential of MCAs as potent vaccine adjuvants, particularly for mucosal vaccines, and summarizes, recent advancements in formulating these activators into nanoparticles to enhance immune responses and protection.

Graphical Abstract

INTRODUCTION

Mucosal Vaccination

A recent study conducted by the World Health Organization (WHO) estimates that within the last 50 years, vaccines have saved at least 154 million lives, with the majority of those saved being infants and small children.(World Health Organization, 2024) Vaccination has proven to be an indispensable means of protecting global health, as it is even responsible for the complete eradication of smallpox, one of the deadliest diseases to ever exist.(Berche, 2022; World Health Organization, 2024) As of January 2025, there are WHO-prequalified vaccines available for 25 human diseases (Samantha Vanderslott, 2022; World Health Organization). Of those 25 diseases, 20 utilize parenteral (intramuscular (I.M.), intradermal (I.D.), or subcutaneous (S.C.)) routes of administration that require a needle. While these vaccines are effective and provide consistent results patient-to-patient, needle-based vaccines are burdened with several significant disadvantages including painful administration, poor compliance due to needle phobias, generation of biohazardous waste, and a lack of production of IgA antibodies in the mucosa.(Gowda et al., 2022) Vaccines for only five of the 25 diseases can be delivered via the mucosal route, with oral vaccines available for cholera, rotavirus, typhoid, and poliomyelitis and an intranasal (I.N.) vaccine for influenza (U.S. Centers for Disease Control and Prevention, 2018, 2019, 2022, 2024a, b). Mucosal vaccines are needle-free, thereby alleviating needle phobias, and importantly can generate strong IgA antibody titers to protect against viral and bacterial infection at mucosal barriers.(Baker et al., 2022) Additionally, FDA recently approved the intranasal influenza vaccine FluMist for at-home use, creating significant market opportunities for needle-free vaccines. Thus, there is an obvious need for the further development of more needle-free mucosal vaccines.

When a parenteral vaccine is administered to a patient, it generates substantial levels of serum IgG antibodies, which are crucial for protecting against pathogens that have entered the systemic circulation and tissues. While IgG and its isotypes are crucial for protection, they are primarily found in the blood and extracellular fluid. While still present in the blood, IgA antibodies are much more common at mucosal surfaces, where they exist in either a soluble or B cell membrane-bound.(Alberts et al., 2002) IgA antibodies are crucial for binding to and neutralize pathogens, preventing their entry into the body.(Janeway Jr et al., 2001) Since an estimated 70-90% of clinically relevant pathogens enter the body through the mucosa (e.g., gastrointestinal, nasal pharyngeal, respiratory, genitourinary tracts), a vaccine that initiates a strong IgA response can be transformative in providing protection across a population.(Fragoso-Saavedra and Vega-López, 2020) The remaining percentage of infections can occur from wounds or penetration of the skin barrier such as through insect bites.(Janeway Jr et al., 2001)

Despite the important role of IgA, parenteral vaccinations are often unable to generate significant concentrations of IgA in the mucosa, allowing pathogens to enter into the body.(Baker et al., 2022) However, some pre-clinical mucosal vaccines have demonstrated that they can generate high concentrations of both serum IgG and mucosal IgA, which makes them ideal for providing robust protection.(Anggraeni et al., 2022; Bento et al., 2015; Hofmann et al., 2009; McLachlan et al., 2008; Ontiveros-Padilla et al., 2023) Due to the constant presence of external antigens, mucosal surfaces are often tolerogenic to prevent unnecessary immune responses to harmless environmental and food-borne antigens.(Alpan et al., 2001) Because of this, adjuvants, compounds that increase the immunogenicity of an antigen, generally need to be included in mucosal vaccine formulations to overcome this tolerogenic barrier.(Baker et al., 2022) Unfortunately, mucosal vaccines are often limited by a lack of available adjuvants that are safe and can be effectively included in the vaccine formulation, hindering additional development. (Table 1.) For instance, cholera toxin is considered a gold standard mucosal adjuvant in animal models, yet it is not safe for use in humans.(Lavelle and Ward, 2022; McLachlan et al., 2008) Thus, new and safe adjuvant options are desperately needed to further progress mucosal vaccine development and mass inoculation.

Table 1.

Advantages and disadvantages of needle and needle-free vaccines

Administration Route	Advantages	Disadvantages
Needle	Strong IgG antibody response	Minor IgA antibody response
		Painful administration
	Widely used	Produces biohazardous waste
	More consistent responses across a population	Requires trained professionals
		Requires extensive cold-chain
	Pre-existing manufacturing infrastructure	Poor patient compliance
Needle-free	Strong IgA antibody response	Lack of available adjuvants
	Bypasses needle phobias	Often requires boosters/poor durability
	Painless administration	Variability between patients
	Can neutralize pathogens at site of entry	Could invoke oral tolerance
		Requires extensive cold-chain
	Immune crosstalk between adjacent mucosa	Short window of opportunity due to intrinsic mucosal defenses

Mast Cells & Mast Cell Activators

Once only thought to be associated with allergies and allergic reactions, mast cells have been identified as playing an important role in a wide range of physiological responses. A type of innate immunity granulocyte, mast cells contain a variety of immunostimulatory mediators such as histamine, heparin, proteases, chemokines, interleukins, and pro-inflammatory cytokines. Upon interacting with a pathogen or allergen, mast cells become activated and degranulate, releasing these immunostimulatory mediators into systemic circulation and activating immune cells such as dendritic cells to migrate to the draining lymph nodes to further mount an immune response.(Johnson-Weaver et al., 2021; Krystel-Whittemore et al., 2015; McLachlan et al., 2008) The surface of mast cells are covered in the FCεRI receptor that has a high-affinity for, and is often naturally bound with IgE antibodies. When the mast cell encounters an antigen, the antigen binds to the IgE antibody and sequentially induces the IgE-FCεRI complexes to cross-link, causing the mast cell to degranulate.(Gaudenzio et al., 2016) (Figure 1.) IgE-independent activation can occur through interactions with a variety of different receptors, such as Mas-related G protein–coupled receptor X2 (MRGPRX2), P2X Purinoceptor 7 (P2RX7), and Adhesion G-Protein-Coupled Receptor E2 (ADGRE2). Activation is also possible via the FcγRs receptor, which behaves similarly to FCεRI, except it has a low-affinity for IgG rather than IgE.(Yang et al., 2023) It is important to note that activation of mast cells does not always result in degranulation, as activated mast cells can also release inflammatory molecules without degranulating.(Theoharides et al., 2007)

Figure 1.

Mast cells that have IgE bound to FCεRI (A) without antigen present and (B) antigen bound to IgE, cross-linking multiple IgE-FCεRI complexes and inducing degranulation.

There are two major types of mast cells present in the body, mast cells that have granules with a combination of tryptase, chymase, and carboxypeptidase (MC_TC), and mast cells with granules that primarily contain tryptase (MC_T). MC_T mast cells are involved in the immune response and are found predominantly in and around mucosal surfaces, while MC_TC mast cells are involved in tissue repair and are located within the submucosa near blood, lymphatic vessels, and connective tissue.(Fong and Crane, 2018) For this review, all mast cell discussion will refer to MC_Ts.

Mast cell activators (MCAs) are a class of predominantly cationic compounds, often small molecules, that are becoming increasingly popular because of their ability to induce mast cell activation and subsequently generate an antigen-specific immune response. Current findings suggest that many therapeutically relevant MCAs induce mast cell degranulation via the G_i-protein dependent receptor: MRGPRX2.(Johnson-Weaver et al., 2021; McNeil et al., 2015; Subramanian et al., 2016) Many MCAs have been found to act as safe and effective adjuvants, especially when compared to several of the available FDA-approved adjuvants.(Staats et al., 2013) The polymer, Compound 48/80 (C48/80), and the antimicrobial peptide, mastoparan-7 (M7), are two of the most common MCA mucosal adjuvant candidates and there have been numerous studies demonstrating their strong efficacy and safety profiles. However, there are several other MCAs such as mastoparan, polymyxin B, β-defensins, and ST101036 that have also demonstrated potential as adjuvants (Table 2).

Table 2.

MCAs and their reported use.

Adjuvant	Disease	Route of Administration	Antigen	Model	Summary
C48/80	Pneumococcal Disease	I.N.	SPY1	Mouse	C48/80 SPY1 vaccinated mice elicited a comparable humoral response to cholera toxin SPY1 vaccinated mice but with a greatly improved safety profile (Zeng et al., 2015)
	Influenza	I.N.	Viral hemagglutinin protein (HA)	Mouse	C48/80 HA vaccinated mice generated substantial levels of IgG, IgA titers and neutralizing antibodies. C48/80 HA vaccinated mice performed similarly to cholera toxin HA vaccinated mice (Meng et al., 2011)
	Pertussis	I.N.	Pertussis B oligomer (PTB)	Mouse	*C48/80 PTB vaccinated mice had higher levels of nasal IgA and serum IgG than mice that were vaccinated with PTB and alum (Hofmann et al., 2009)
	Botulism	I.N.	Recombinant botulinum neurotoxin type A heavy chain β-trefoil domain fused with an adenovirus type 2 fiber protein (Hcβtre-Ad2F)	Rabbit	C48/80 Hcβtre-Ad2F vaccinated rabbits produced strong levels of serum IgG titers and neutralizing antibody titers that were comparable to those generated by cholera toxin Hcβtre-Ad2F vaccinated rabbits (Staats et al., 2011)
	Anthrax	I.D.	Bacillus anthracis protective antigen (PA)	Mouse	*C48/80 PA vaccinated mice perform similarly to CpG PA and cholera toxin PA vaccinated mice (McGowen et al., 2009)
	Anthrax	I.N.	Bacillus anthracis protective antigen (PA)	Mouse	C48/80 loaded nanoparticles with PA generated significantly greater IgG and IgA antibody titers compared to PA alone (Bento et al., 2015)
	Anthrax	I.N.	Bacillus anthracis protective antigen (PA)	Mouse	C48/80 PA vaccinated mice produced significantly greater titers of saliva, vaginal, and fecal IgA than cholera toxin PA vaccinated mice (McLachlan et al., 2008)
M7	Cocaine abuse	I.N.	Cocaine or GNC analog	Mouse	*M7 cocaine/GNC analog vaccinated mice were protected against the psychoactive effects of cocaine and had lower brain concentrations of cocaine metabolites compared to naïve mice (St. John et al., 2020)
	Influenza	I.N.	COBRA for HA protein	Mouse	*M7-CpG nanoparticles with HA vaccinated mice generated high titers of serum IgG and were 100% protected from a lethal influenza challenge (Ontiveros-Padilla et al., 2023)
ST101036	West Nile Fever	I.M.	West Nile Virus Envelope III protein (EDIII)	Mouse	*Ace-DEX microparticles encapsulating ST101036 with EDIII vaccinated mice produced greater IgG, IgG1, and IgG2 antibodies than EDIII alone and also provided a 90% survival rate when challenged with a lethal dose of West Nile Virus (Hendy et al., 2023)

^*= Direct comparison with FDA-approved adjuvants

Compound 48/80 (C48/80)

First described in 1951, C48/80 is a mixed polymer of p-methoxy-N methyl phenylethylamine crosslinked by formaldehyde that was found to initiate mast cell degranulation.(Paton, 1951; Schemann et al., 2012) McLachlan et al., first observed that when both wild-type and mast cell-deficient mice received C48/80 and Bacillus anthracis protective antigen (PA) I.D. in the footpad, there were significant PA-specific IgG titers in the wild-type mice but not the mast cell-deficient mice; demonstrating both the importance of mast cells for generating protective responses against invading pathogens, and C48/80’s adjuvanticity. Additionally, when mice I.N. received C48/80 and PA there was an increased PA-specific cellular responses compared to mice that received PA alone. This suggests that C48/80 is responsible for activating mast cells found in the nasal associated lymphoid tissue (NALT). To evaluate C48/80’s potential as a mucosal vaccine, McLachlan et al., compared C48/80 to mice I.N. vaccinated with cholera toxin and found that mice which received C48/80 demonstrated significantly greater PA-specific IgG titers, as well as greater saliva, vaginal and fecal derived-IgA titers than the mice that received cholera toxin. (McLachlan et al., 2008) There have been several other studies that suggest C48/80 performs similarly to cholera toxin, but with an improved safety profile.(McGowen et al., 2009; Meng et al., 2011; Zeng et al., 2015; Zheng et al., 2015)

To assess the ability of C48/80 to protect young mice against a Streptococcus pneumoniae infection, Zeng et al., I.N. vaccinated mice with C48/80 and the attenuated pneumococcal strain D39 (SPY1) and compared it to mice that received SPY1 with cholera toxin or the TLR2/TLR6 agonist Pam2CSK4. To assess the presence of long-term antibody titers, serum IgG was measured at 1 and 12 weeks after the last vaccination, with titers remaining high for both cholera toxin and C48/80, but not Pam2CSK4 (Figure 2A). Interestingly, C48/80 was the only adjuvant to maintain significant levels of IgG titers in nasal wash (Figure 2B). However, only cholera toxin maintained long-term saliva and nasal wash IgA levels out to 12 weeks after the last vaccination (Figure 2C–D). The adjuvants were compared in a pneumococcal infection model and mice that received SPY1 + C48/80 had a decreased presence of pneumococcal colonization compared to SPY1 alone, SPY1 + cholera toxin, and SPY1 + Pam2CSK4. When compared in a lethal infection model, there was a 100% survival rate for mice that had received SPY1+C48/80, while mice that received SPY1 + cholera toxin or SPY1 + Pam2CSK4 had a 90% and 70% survival rate, respectively (Figure 2E). Overall, because C48/80 elicited a comparable humoral response, maintained superior resistance to pneumococcal colonization, and provided 100% survival from pneumococcal infection, the study suggested that C48/80 was superior to both Pam2CSK4 and cholera toxin. Additionally, these factors also correlated with the increased protection that C48/80 provides against pneumococcal infection.(Zeng et al., 2015)

Figure 2.

Long-term SPY1-specific antibody titers from mice immunized with the different adjuvants. (A) Serum IgG, (B) Nasal Wash (NW) IgG, (C) saliva IgA, and (D) NW IgA (E) Survival curve of mice that were vaccinated with the different adjuvants, with or without SPY1, and challenged with a lethal dose of pneumococcal. N.D. = No Data, CT = cholera toxin. *P < 0.05, **P < 0.01 (Zeng et al., 2015) Reprinted with permission.

In a study assessing C48/80’s adjuvanticity against H1N1 influenza virus, Zheng et al. found that mice I.N. immunized with a combination of viral nucleoprotein and C48/80 were provided complete protection from a lethal challenge of H1N1 virus, as well as the added benefit of having cross-protection from the H9N2 avian influenza virus. Meanwhile, mice that received the same dose of protein antigen and C48/80 via the intraperitoneal (I.P.) route were not protected against the lethal H1N1 challenge. While there were comparable serum IgG titers between groups that received the same doses of protein antigen and C48/80 adjuvant via either I.N. or I.P. administration, only the I.N. group was able to survive the lethal challenge, demonstrating the effectiveness of C48/80 as a mucosal adjuvant. Concurrently, groups that were I.N. vaccinated were found to have high levels of IgA in the nasal mucus, while I.P. vaccinated groups had undetectable levels. This suggests that the I.N. administration and subsequent development of IgA was crucial for survival against influenza. The protein antigen and C48/80 group also produced significantly higher levels of the pro-inflammatory cytokine, IFN-γ, than the protein antigen group alone did, reinforcing that C48/80 acts as a powerful mucosal vaccine adjuvant.(Zheng et al., 2015)

In a similar study, Meng et al., administered mice I.N. with C48/80 and several increasing doses of recombinant viral hemagglutinin (HA) protein, as well as a cholera toxin and HA protein as a positive control. The C48/80 adjuvanted groups produced serum HA-specific IgG in a dose-dependent manner, with the results from the highest dose group reporting comparable IgG to the cholera toxin group. In a separate experiment, mice were I.N. vaccinated with either HA alone, C48/80, or with cholera toxin to further compare the differences between the two adjuvants. Comparable titers of serum HA-specific IgG and HA-specific IgA in vaginal, lung, and nasal washes and in fecal extract were observed between both adjuvants, with significantly lower titers identified in the PBS and HA alone groups. Adjuvanted groups provided significantly higher levels of neutralizing antibodies than the HA alone group when tested against the influenza strain, A/California/04/2009. This same live virus was used in a challenge model, where 100% of the cholera toxin group and 90% of the C48/80 groups survived an influenza challenge after being vaccinated with HA and the respective adjuvant. The adjuvanted mice that survived the challenge had only minor damage to lung tissue and extremely low viral load, compared to the PBS and HA alone mice which had severe lung damage and a high viral load. The results of this study show that C48/80 has a similar effectiveness to cholera toxin.(Meng et al., 2011)

In a small study, Hofmann et al., assessed C48/80’s adjuvanticity in an I.N. vaccine against the highly infectious bacteria, Bordetella pertussis, the causative agent of Pertussis (Whooping Cough). Mice were vaccinated intranasally (I.N.) with pertussis B oligomer (PTB), a subunit of the pertussis toxin, along with C48/80. These mice were then compared to those that received either I.N. PTB alone or a subcutaneous (S.C.) injection of PTB with the FDA-approved adjuvant, aluminum hydroxide (alum). The mice that received C48/80 had significantly higher levels of IgA in their nasal secretions compared to both the unadjuvanted or alum group, as well as significantly higher and comparable levels of serum IgG to the unadjuvanted or alum group, respectively. This study demonstrates the unique adjuvanticity of I.N. administered C48/80. (Hofmann et al., 2009)

While the importance of effective adjuvants cannot be understated, Staats et al., took a unique approach by developing a recombinant antigen for a Clostridium botulinum vaccine that also included C48/80 as an adjuvant. C. botulinum is an infectious bacterium that is responsible for producing botulinum neurotoxin, considered to be the deadliest substance on Earth.(Dhaked et al., 2010; Dunbar, 1990) The recombinant antigen consisted of the botulinum neurotoxin type A heavy chain β-trefoil domain fused with an adenovirus type 2 fiber protein (Hcβtre-Ad2F). Ad2F was included in the antigen formulation to act as a mucosal targeting ligand. The recombinant antigen was combined with either cholera toxin or C48/80 and used to vaccinate I.N. New Zealand White Rabbits. Both the cholera toxin and C48/80 adjuvanted groups generated Hcβtre-Ad2F specific IgG antibody titers, with the former producing the highest titers between the two groups. Unadjuvanted Hcβtre-Ad2F was unable to generate any IgG. Interestingly, rabbits that received Hcβtre with either adjuvant, but without Ad2f produced virtually no titers, demonstrating the importance of utilizing the mucosal targeting ligand. The experiment was repeated with a different rabbit strain, Dutch Belted Rabbits, to ensure that the experimental results could be replicated in a different population, with similar results being observed. Additionally, both adjuvant groups produced comparable levels of neutralizing antibodies against botulinum toxin A, while the Hcβtre and Hcβtre-Ad2F alone groups could not.(Staats et al., 2011)

McGowen et al., I.D. vaccinated mice via the ear pinnae with PA and either C48/80, cholera toxin, or the FDA-approved synthetic oligonucleotide, cytosine phosphate guanine (CpG), at varying doses (3, 10, and 30 μg) and found that 30 μg of C48/80 generated protective serum PA-specific IgG antibodies that were comparable to those produced by mice that had received PA with either doses of cholera toxin (0.1 and 1 μg) or CpG (1 and 10 μg) (Figure 3A). Additionally, the 30 μg dose of C48/80 and either dose of cholera toxin or CpG were found to generate comparable levels of functional neutralizing antibodies against anthrax lethal toxin, suggesting that any of the adjuvants would provide adequate protection against a B. anthracis challenge (Figure 3B). To assess the cellular response differences between the adjuvants, spleens from each group were harvested to measure the different cytokines produced with antigen recall. The greatest dose of each adjuvant produced statistically comparable levels of IL-4, IL-6, IL-17, and IFN-γ to be produced by the splenocytes. While this study did not utilize a mucosal vaccination strategy, C48/80 was shown to perform just as well as cholera toxin and CpG as an adjuvant, albeit at a much larger dose of C48/80.(McGowen et al., 2009)

Figure 3.

(A) Geometric mean titers (GMT) of PA-specific serum IgG produced by varying doses of each adjuvant. (B) The 75% neutralization titer (NT75) produced by varying doses of each adjuvant. rPA = recombinant PA. *p < 0.05, **p < 0.01, ***p < 0.001 (McGowen et al., 2009) Reprinted with permission.

Mastoparan and M7

Mastoparan is an antimicrobial small molecule peptide that is derived from the venom of wasps and honeybees. It was initially isolated from the venom sacs of Korean yellow-jacket wasps via column chromatography and was found to possess mast cell activating properties after it was applied to rat peritoneal mast cells which subsequently released histamine.(Hirai et al., 1979) It was later observed in the macrophage cell line, J774, that mastoparan could induce the release of prostaglandin E₂, which is found in mast cell granules. Additionally, mastoparan was found to act as a weak adjuvant by eliciting a Th2-biased immune response in mice.(King et al., 2003) However, more recently, mastoparan-7 (M7), an analog of mastoparan, has been reported to act as a more potent MCA.(St. John et al., 2020) Based on the current available literature, M7 appears to be more commonly used than mastoparan (Table 1).

To combat the use of the illicit drug cocaine, St. John et al., developed a cocaine vaccine using M7 as an adjuvant. Because cocaine is a small molecule, it is unable to stimulate the immune system on its own, preventing it from generating cocaine-specific antibodies. To circumvent this issue, small molecules can be conjugated to larger and more immunogenic carrier proteins that ultimately improve the small molecule-specific immune response. Small molecules conjugated to carrier proteins in this manner are referred to as haptens. St. John et al., conjugated either cocaine or its analog, 6-(2R,3S)-3- (benzoyloxy)-8-methyl-8-azabicyclo [3.2.1] octane-2-carbonyloxy-hexanoic acid (GNC), to the protein carrier, keyhole limpet hemocyanin (KLH) and combined it with M7 for I.N. vaccination. This formulation was compared against I.P. administered alum and S.C. administered alum followed by an I.N. boost with M7. The cocaine and GNC groups that received alum I.P., or alum S.C. followed by an I.N. boost with M7 had significant and comparable serum hapten-specific IgG titers compared to the group that received the hapten I.N. administered alone. The group that received M7 I.N. without a S.C. alum prime performed well, but not as well as the group with the S.C. alum prime. Mice were later challenged with cocaine to assess the efficacy of the different haptens, adjuvants, and routes of administration, via increased movement associated with the psychoactive effects of the drug. Despite the high titers generated by the groups that received alum I.P., or alum S.C. followed by an I.N. boost with M7, neither group was able to prevent or even reduce the psychoactive effects of cocaine. Meanwhile, the group that received cocaine-KLH combined with M7 I.N. significantly reduced the amount of mouse movements compared to the naive group and the groups that had received alum. Additionally, the main cocaine metabolite present in the brain, norcocaine, was measured via mass spectrometry and only the groups that had received either cocaine-KLH with M7 I.N. or GNC-KLH with alum had significantly lower brain concentrations than the naive group. It was also observed that the mice that received I.N. M7 from either cocaine-KLH or GNC-KLH had much higher saliva IgA levels than the naive group or those that received alum I.P (Figure 4A–B). It is interesting to note the variability in cocaine and GNC-specific IgA titers between groups, as this could be due to slight molecular differences between cocaine and GNC. Because cocaine is typically self-administered nasally, the development of an I.N. vaccine formulation that generates high levels of cocaine-specific IgA antibodies is ideal. This study demonstrated that M7 was successfully incorporated as a mucosal vaccine adjuvant and that it was able to perform similarly to alum as a potential means for fighting chronic cocaine abuse.(St. John et al., 2020)

Figure 4.

Saliva IgA titers of (A) cocaine-specific and (B) GNC-specific antibodies.(St. John et al., 2020). Reprinted with permission.

Polymyxin B

Polymyxin B is an FDA-approved antibiotic used for treating gram-negative bacterial infections.(Shatri and Tadi, 2020) Polymyxin B was originally found to activate mast cells after it was S.C. administered to rats, and similar side effects were observed as caused by C48/80. Surprisingly, polymyxin B activated mast cells more strongly than C48/80 while still maintaining low levels of toxicity.(Bushby and Green, 1955; Parratt and West, 1957) Yoshino et al., demonstrated that after mice were I.N. vaccinated with the model antigen, ovalbumin (OVA), or OVA with either polymyxin B or the antibiotic, colistin, there was increased OVA-specific IgA in fecal extracts, nasal washes, vaginal washes, saliva, and plasma while also increased OVA-specific plasma IgG with increasing dose compared to mice that received OVA alone. Notably, mice that received OVA alone did not generate any OVA-specific IgA in fecal extracts, nasal washes, or saliva. Cell suspensions of lymph nodes (LNs), NALT, lamina propria (LP), and spleens were characterized to assess the quantity of OVA-specific antibody forming cells. There were statistically significant increases in cells producing OVA-specific IgA in samples of LP and the spleen and OVA-specific IgG in samples of LP, LNs, and the spleens of mice immunized with either polymyxin B or colistin compared to mice immunized with OVA alone. The authors noted that OVA-specific IgA and IgG antibodies began to decline after several weeks, but after the administration of an I.N. boost, there was a substantial increase in plasma IgG and IgA antibodies that remained elevated for 31 weeks. The addition of the boost also elevated the levels of cells producing OVA-specific IgA in samples of LP and OVA-specific IgG in samples of LP and spleens.(Yoshino et al., 2013) Despite the potent mast cell activating properties, there are relatively few examples of polymyxin B being used as a vaccine adjuvant, suggesting more research is needed.

β-defensins

Defensins are a type of endogenous antimicrobial peptide that are highly conserved across many organisms.(Semple and Dorin, 2012) More specifically, β-defensins, a subfamily of defensins, are known to activate mast cells through the MRGX2 receptor.(Choi et al., 2019; Subramanian et al., 2013) While recognized as MCAs, β-defensins are often predominantly associated with their chemoattractant abilities on various immune cells, primarily CD4+ T cells.(Biragyn et al., 2002; Niyonsaba et al., 2004; Shelley et al., 2020). As mentioned previously, there are few sources that describe the use of β-defensins as mucosal adjuvants, but rather explore their ability to induce dendritic cell maturation and migration towards lymph nodes, which can result in improved antigen-specific immune responses.(Biragyn et al., 2002; Biragyn et al., 2001) Tani et al., observed that mice which received I.P. KLH adsorbed to alum with daily injections of defensins increased the number of KLH-specific IgG, IgG1, and IgG2b antibodies compared to mice that did not have the daily defensin injections. The mice that received daily injections of defensins also had increased levels of mature B cells and IFN-γ expression.(Tani et al., 2000) While β-defensin shows promise, it is evident that further studies are much needed to evaluate its efficacy as a mucosal adjuvant.

The Need for Improved Formulations

While specific mucosal physiological conditions differ based on the location in the body, the mucosal membrane is a major barrier for efficient delivery of drugs and vaccines.(Leal et al., 2017) One of the fundamental roles of the mucosal membrane is to quickly and effectively remove pathogens and debris, thus, it is naturally efficient at preventing administered therapies from localizing and eliciting a response. Mucosal membranes possess various mechanisms to prevent access to the underlying cellular epithelium. These include a complex array of mucins and other biological molecules within the mucus that confer high viscoelasticity, diverse hydrophobic and ionic interactions, and the capability to filter out large particles.(Leal et al., 2017) While the pH of mucus varies based on location, the nasal mucosa is slightly acidic, ranging from 5.5-6.7.(Leal et al., 2017; Zhao et al., 2022) Due to these challenges, unique formulations are required that enable improved penetration of the mucosal membrane.

One formulation that has potential to enhance delivery to the mucosa is nanoparticles. Nanoparticle formulations are becoming increasingly more common, as seen with the recent development and distribution of the COVID-19 lipid nanoparticle mRNA vaccines. There are numerous advantages to using nanoparticle-based formulations, such as controlled antigen delivery and presentation, improved circulation time, and dose sparing in that smaller quantities of antigen and adjuvant are required compared to soluble delivery.(Bezbaruah et al., 2022; Lozano et al., 2023) The highly modifiable physicochemical properties of nanoparticles allow for many combinations of varying sizes, surface charges, and chemical compositions that can be tailored to specific needs. For mucosal vaccines, nanoparticles can be modified to have highly mucoadhesive properties, enabling them to efficiently bind to and be retained by mucus. This would allow the nanoparticles to subvert the mucosa’s efficient clearance mechanisms and ensure that an effective dose of vaccine is delivered.(Shaikh et al., 2011; Zhao et al., 2022)

The widely used hydrophilic and biocompatible polymer, polyethylene glycol (PEG), is frequently included in mucosal nanoparticle formulations because of its mucopenetrating properties. Low molecular weight PEG is known to enhance mucosal penetration by greatly reducing the interactions between nanoparticles and mucins, enabling improved movement throughout the mucosa.(Huckaby and Lai, 2018) Regardless of its widespread use, there are concerns that PEG may invoke an immune response when it is bound to carriers. These immune responses often result in the development of anti-PEG antibodies that can lead to anaphylaxis, or even the inactivation of other medications containing PEG.(Ibrahim et al., 2022; Kozma et al., 2020; Vasquez-Martínez et al., 2023) Although still a concern, PEG-induced immune responses are a relatively new area of study that requires additional research. While the advantages of using a carrier system for MCA delivery to the mucosa are numerous, there are few experimental examples of MCA formulation being considered.

Formulated MCAs in vaccines

Compound 48/80 (C48/80)

Bento et al., investigated whether chitosan, a cationic, biodegradable polymer, or alginate, an anionic, natural, biodegradable polymer, combined with chitosan could be used to create nanoparticles and encapsulate C48/80. Interestingly, because of its’ positive charge, chitosan is inherently mucoadhesive, as it binds to the negatively charged mucins within the mucus.(M. Ways et al., 2018) Through a simple formulation process, C48/80 was encapsulated in both chitosan and chitosan/alginate positively charged nanoparticles (Figure 5A). To assess the cytotoxicity of the nanoparticle formulations, primary mouse splenocytes and the cancerous human lung cell line, A549, were treated with the different nanoparticles as well as soluble C48/80. It was found that there was an increase in cell death with an increase in soluble C48/80 concentration, yet when C48/80 was encapsulated in either nanoparticle there was a decrease in cytotoxicity compared to the soluble form, demonstrating the effectiveness of utilizing nanoparticles to minimize cytotoxicity. To assess the in vivo effects, the nanoparticles had B. anthracis PA adsorbed to their surface and were administered to mice I.N., where it was found that the nanoparticles generated strong levels of PA-specific IgG titers that were significantly higher than groups that received PA alone (Figure 5B). The combination of C48/80 with chitosan created a balanced Th1/Th2 immune response, as seen through comparable PA-specific IgG1 and IgG2c antibody titers (Figure 5C). However, the chitosan/alginate-C48/80 nanoparticles were slightly outperformed by the chitosan-C48/80 nanoparticles. Mucosal immunity was also improved when compared to PA alone, as C48/80 encapsulated chitosan nanoparticles generated significantly higher PA-specific nasal, vaginal, and fecal IgA titers. It is interesting that chitosan/alginate-C48/80 nanoparticles did not perform as well as the chitosan-C48/80 nanoparticles, as chitosan and alginate have previously been indicated to act as an agonist and up regulator of TLR4, respectively.(Hoffman et al., 2023) It is plausible that there could be slight antagonistic interactions occurring between the three polymers, or that chitosan and alginate are somehow limiting the full effect of C48/80. It would be beneficial for future efforts to investigate these interactions in order to develop an optimized formulation. Overall, this study demonstrated that formulating C48/80 through the development of a nanoparticle delivery system lead to an effective vaccine.(Bento et al., 2015)

Figure 5.

(A) Size, polydispersity index (PI), zeta potential (ZP), and loading efficiency (LE) of the different nanoparticle formulations. (B) Geometric mean titers (GMT) of serum PA-specific IgG over 42 days. (C) Day 42 serum PA-specific IgG1 and IgG2c. Chi-C48/80 NP = Chitosan-C48/80 nanoparticles, Chi NP = Chitosan nanoparticles, Chi/Alg-C48/80 NP = Chitosan/Alginate-C48/80 nanoparticles, Chi/Alg NP = Chitosan/Alginate nanoparticles. * p < 0.05, ** p < 0.01, *** p < 0.001 (Bento et al., 2015) Reprinted with permission.

While many studies focusing on I.N. vaccines tend to utilize liquid formulations, Wang et al., developed a dry powder formulation for an anthrax vaccine, incorporating C48/80 and PA. Spray-Freeze-Drying (SFD) was used to produce several different porous microparticle formulations consisting of varying quantities of C48/80, PA, and the cryoprotectant, trehalose (Figure 6A–C). As part of the study, rabbits were I.N. vaccinated with either the powder formulation via a unit dose powder inhaler or a positive control liquid formulation and compared to rabbits who received a I.M. injection. To ensure that the liquid formulations were retained in the nasal cavity, rabbits were left on their backs for 30 seconds post immunization. The powder, liquid, and I.M. formulations produced significantly higher anti-PA IgG antibodies than PA did alone. However, the powder and I.M. formulations generated measurable antigen specific IgG titers after only one dose, while the liquid formulations did not. After three doses of the vaccine were administered, all formulations performed similarly and generated exceptionally higher serum IgG titers compared to the unadjuvanted PA group. Additionally, all adjuvanted formulations generated substantial neutralizing antibodies against anthrax lethal toxin, while the unadjuvanted PA did not. The encapsulation of C48/80 and PA into porous microparticles resulted in significantly improved immune response and efficacy compared to unformulated PA, once again showcasing how improving the formulation of a compound or therapy can greatly increase its efficacy (Wang et al., 2012)

Figure 6.

Scanning electron microscope (SEM) images of (A) SFD trehalose C48/80 formulation (B) SFD rPA formulation-1 and (C) SFD rPA formulation-2. Scale bars represent 10 μm, 20 μm, and 10 μm, respectively. rPA = recombinant PA (Wang et al., 2012) Reprinted with permission.

M7

Since MCAs are primarily cationic, they can be formulated with anionic molecules to form complexes. Ontiveros-Padilla et al., was able to formulate nanoparticle complexes between M7 and CpG, based on the ratio of the positively charged amine groups of M7 and the negatively charged phosphate groups of CpG via sonification (Figure 7A). These complexes demonstrated comparable cytotoxicity to M7 or CpG alone in a murine dendritic cell line, DC2.4, and a murine mast cell line, MC/9, but had decreased cytotoxicity in LA-4 cells, a murine lung epithelial cell line. (Figure 7B–D). The M7-CpG complexes were administered I.N. in mice with a computationally optimized broadly reactive antigen (COBRA) for the influenza HA protein. It was observed that the M7-CpG complexes elicited high COBRA-specific serum IgG titers compared to unadjuvanted groups, as well as a significant difference between groups that used only one of the two adjuvants. The mice that received the M7-CpG complexes had greater quantities of IL-2 and IFN-γ producing splenocytes after antigen recall, and overall, more IFN-γ was produced than cells isolated from mice in the unadjuvanted and single adjuvant groups. However, the mice that received only M7 produced greater amounts of IL-4 after restimulation with antigen than the mice that received M7-CpG complexes, but the M7-CpG vaccinated mice still produced significantly more than the CpG or unadjuvanted groups. This indicated that the M7 invoked a more skewed Th2 response compared to Th1 skewing CpG. Complexation of the two adjuvants resulted in a balanced Th1/Th2 response, which can be ideal for a vaccine, offering a broadly protective immune response. To evaluate the overall protection provided by each adjuvant, mice were challenged with a mouse-adapted variation of the influenza strain, A/Hong Kong/1/1968. The M7-CpG complex group and CpG group were provided 100% and 70% protection, respectively, and only minor weight loss, while the PBS, unadjuvanted, and M7 group all succumbed to the virus. This study demonstrated the efficacy of formulated adjuvant complexes over individual soluble adjuvants, as well as the advantages of vaccines that utilize different combinations of adjuvants in tandem.(Ontiveros-Padilla et al., 2023)

Figure 7.

(A) Transmission electron microscopy (TEM) image of the M7-CpG complexes. (B) Percent LDH release in DC2.4 cells, (C) MC/9 cells, (D) LA-4 cells. Scale bar represents 1 μm. *p-value ≤ 0.05 (Ontiveros-Padilla et al., 2023) Reprinted with permission.

Small molecule ST101036

The small molecule, 1-[6-(4-ethylphenyl)-4-(trifluoromethyl)pyrimidin-2-yl]-4-piperidylamine (ST101036), was identified as a MCA through a high-throughput screening analysis of over 55,000 different compounds. Each compound was added to MC/9 cells and the subsequent release of β-hexosaminidase was measured, as β-hexosaminidase is a granular component of mast cells. ST101036 was found to be one of several potent MCAs identified.(Choi et al., 2019) However, ST101036 is poorly soluble in aqueous solutions, so the use of a delivery vehicle is required to ensure its’ effectiveness in vivo. Hendy et al. provided an analysis of the use of acetalated dextran (Ace-DEX) microparticles to encapsulate ST101036. Ace-DEX is a biocompatible, highly tunable, acid sensitive, hydrophobic polymer that has demonstrated its efficacy as an accomplished vaccine delivery system.(Bachelder et al., 2017) Through nanoprecipitation, the Ace-DEX microparticles were able to successfully encapsulate ST101036 with an efficiency of 93.74%, maximizing the quantity of MCA that can be delivered (Figure 8A–B). The efficacy of the ST101036-encapsulated Ace-DEX microparticles was tested in an infection model using West Nile Virus, where ST101036 microparticles were combined with soluble West Nile Virus Envelope III protein (EDIII) and used to vaccinate mice S.C. The ST101036 microparticles were compared to soluble EDIII alone, blank microparticles, the commonly used adjuvant, alum, and M7 as a positive control group. ST101036 microparticles produced greater anti-EDIII IgG titers compared to the soluble EDIII group while also exhibiting comparable results to the alum group. Similarly, ST101036 microparticles produced much higher IgG1 and IgG2 antibodies compared to the soluble EDIII group (Figure 8C). The mice were subsequently challenged with a lethal dose of West Nile Virus, and the ST101036 microparticle group exhibited an impressive overall survival rate of 90%, whereas the soluble EDIII group had a meager survival rate of only 50% (Figure 8D). This study highlights the effectiveness of Ace-DEX microparticles as a vehicle for vaccine delivery and shows how formulation promotes the efficient delivery and application of MCAs.(Hendy et al., 2023)

Figure 8.

Scanning Electron Microscopy (SEM) images of (A) Blank Ace-DEX nanoparticles (NP) and (B) ST101036 loaded Ace-DEX microparticles (MP). (C) Day 42 IgG, IgG1, and IgG2a titers produced by soluble EDIII, blank MPs, and each adjuvant. (D) Survival curve of mice that were vaccinated with the different adjuvants with soluble EDIII and challenged with a lethal dose of West Nile Virus. Scale bars represent 1 μm. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001. (Hendy et al., 2023) Reprinted with permission.

POTENTIAL CONCERNS OVER THE USE OF MAST CELL ACTIVATORS

While MCAs have great potential for use as adjuvants, there are rightfully some concerns regarding their safety. The intention of using MCAs as adjuvants is to induce localized mast cell activation yet concerns stem from the fact that poorly characterized MCAs could potentially cause systemic mast cell activation, leading to anaphylaxis. Anaphylaxis is characterized by a hypersensitive reaction to a specific antigen that can result in life-threatening side effects or even death.(Reber et al., 2017) This reaction is caused in part by systemic mast cell activation and degranulation that results in the body being quickly flooded with granular contents from the mast cells. While there are a wide variety of proteins, proteases, lipids, and pro-inflammatory cytokines released, one of the most consequential molecules is histamine. Numerous studies have demonstrated the major role that histamine plays in the onset of anaphylaxis, as it is a potent vasodilator.(Reber et al., 2017) The release of significant quantities of histamine, as well as other immunostimulatory molecules, induces vasodilation that leads to hypotension. If the hypotension is not quickly alleviated, then it can lead to hypoperfusion which could result in death if left untreated.(Ramanlal and Gupta, 2020)

While anaphylaxis is predominantly induced in an IgE-dependent manner, IgE-independent anaphylaxis is possible, although it is thought to be much less common (Gülen and Akin, 2022; Reber et al., 2017). Assessing serum IgE titers is a means of experimentally identifying early warning signs of anaphylaxis post vaccination. One of the reasons that M7 may be favored over mastoparan is that mastoparan was observed to have an effect on increasing the production of IgE titers, leading to a questionable safety profile.(King et al., 2003). Zeng et al., performed an in-depth analysis of both C48/80 and cholera toxin to compare the relative safety of each. Young mice I.N. received either C48/80 or cholera toxin for seven days, and any subsequent changes in body weight, serum IgE, pro-inflammatory cytokines, and organ pathology were measured. Mice that received C48/80 saw normal increases in body weight, while mice that received cholera toxin lost weight and struggled to regain it (Figure 9A). There was no indication of C48/80 induced anaphylaxis, as serum IgE levels from C48/80 were comparable to that of PBS, while cholera toxin induced high levels of IgE (Figure 9B). The inflammatory cytokines IL-6 and IL-1β were also higher in the nasal wash of the cholera toxin group while the C48/80 group was again comparable to PBS. There was no statistical difference in TNF-α levels between each group (Figure 9C–E). Finally, H&E staining was performed on the brains and lungs of each group. Aside from slight leukocyte infiltration in the lungs, C48/80 was comparable to the PBS group and there was no observable pathology in the brain. The lungs of the cholera toxin group had destroyed pulmonary alveoli and induced neuronal necrosis in the brain. These results indicate that C48/80 is significantly safer than cholera toxin.(Zeng et al., 2015)

Figure 9.

(A) Change in mouse body weight after receiving each adjuvant. (B) Serum IgE titers of each group. Nasal wash levels of the pro-inflammatory cytokines (C) TNF-α, (D) IL-6, and (E) IL-1β. NW = nasal wash. *P < 0.05, **P < 0.01 (Zeng et al., 2015) Reprinted with permission.

McGowen et al., had a similar observation, as neither C48/80 nor CpG generated antigen-specific IgE, while cholera toxin did. Interestingly, Meng et al., noted that they found no discernible difference in serum IgE levels between C48/80, cholera toxin, or HA protein. The authors note that despite the lack of a difference between the three groups, there was no HA-specific IgE observed in any of the groups, implying that C48/80 is not associated with IgE production. Additionally, it should be noted that there are differences in the route of administration and doses of C48/80 and cholera toxin between studies which could likely account for variations in the measured IgE concentrations. While not directly comparing to cholera toxin, Yoshino et al., found that there was no significant difference between serum IgE levels generated by mice that received OVA alone or OVA with polymyxin B, suggesting there is minimal risk of polymyxin B inducing anaphylaxis. A disadvantage of monitoring IgE titers is that it is measured via an enzyme-linked immunosorbent assay (ELISA) that takes several hours to see results. If an animal model were to develop anaphylaxis, the clinical signs would become obvious much earlier than the ELISA results.

Evaluating body temperature post vaccination is one of the least labor-intensive means of more rapidly identifying a potential anaphylactic response. One of the main side effects of anaphylaxis is a large decrease in body temperature, which is due to thermo-responsive neurons becoming activated from degranulating mast cells that then trick the brain into believing that the body is overheating, thus a subsequent decrease in body temperature follows.(Bao et al., 2023; Gouel-Chéron et al., 2023) It was observed in mice that a decrease in body temperature can precede any obvious clinical signs of anaphylaxis, allowing researchers to quickly intervene if necessary.(La Rotta et al., 2009)

Finally, the use of Collaborative Cross (CC) mice as a means of evaluating safety could be greatly informative for further studies involving MCAs. CC mice are a recombinantly inbred strain of mice that are derived from 8 different ancestorial founder strains comprising a wide breadth of genetic variation. These mice more accurately reflect the immense immunologic diversity found throughout the human population compared to commonly used C57BL/6 and BALB/c strains.(Threadgill et al., 2011) CC mice have been found to possess varied mast cell activity and IgE sensitivity across different strains. In particular, the strain, CC027, was identified as possessing increased mast cell sensitivity and is capable of eliciting strong Th2-skewed immune responses that are associated with enhanced mast cell activity. (Matsushita et al., 2021) Furthermore, the CC027 strain have been used to assess oral tolerance in relation to peanut allergies, a condition in which human responses range from mild to severe and in which mast cells play a predominant role.(Orgel et al., 2019) The genetic variation found within CC mice can be utilized to develop an improved understanding of the safety profile for pre-clinical MCAs, as some mice may have more dramatic immune responses compared to others, mimicking the human population and enabling further development and modification if necessary.

CONCLUSION

The constant evolving threat of infectious diseases and the devastation that they can cause is an important reminder of the need for effective vaccines. To further safeguard public health, the desire for greater accessibility and easier to administer vaccines is leading to more attention being placed on needle-free alternatives like mucosal vaccines. Despite the many advantages that mucosal vaccination offers, it is still often necessary to include adjuvants in the vaccine formulations. As described in this review, the use of MCAs as adjuvants provides a good means of increasing the effectiveness of mucosal vaccines. There is a wealth of evidence suggesting that MCA adjuvants are safer than the current gold standard mucosal vaccine adjuvants and perform similarly to FDA-approved adjuvants. It is apparent that the formulation of MCAs can lead to enhanced safety and effectiveness, as nanoparticle-based delivery systems can be utilized to develop impressive vehicles for MCA delivery. While the future for MCA-incorporated mucosal vaccine formulations is promising, there is still an abundance of research necessary for the further development and validation of MCAs as vaccine adjuvants.