Induction of protective immune responses at respiratory mucosal sites

ABSTRACT

Many pathogens enter the host through mucosal sites. Thus, interfering with pathogen entry through local neutralization at mucosal sites therefore is an effective strategy for preventing disease. Mucosally administered vaccines have the potential to induce protective immune responses at mucosal sites. This manuscript delves into some of the latest developments in mucosal vaccination, particularly focusing on advancements in adjuvant technologies and the role of these adjuvants in enhancing vaccine efficacy against respiratory pathogens. It highlights the anatomical and immunological complexities of the respiratory mucosal immune system, emphasizing the significance of mucosal secretory IgA and tissue-resident memory T cells in local immune responses. We further discuss the differences between immune responses induced through traditional parenteral vaccination approaches vs. mucosal administration strategies, and explore the protective advantages offered by immunization through mucosal routes.

KEYWORDS:

• Mucosal
• respiratory tract
• vaccine
• adjuvant
• virus
• vaccinology

Introduction

Mucosal surfaces, which line the respiratory, digestive, and genitourinary tracts, serve as primary barriers against environmental factors such as pathogens and allergens. These surfaces are continuously monitored by the immune system, balancing tolerance to benign factors and activation against harmful microbes. Traditional vaccines, administered subcutaneously or intramuscularly, primarily elicit systemic immune responses with elevated levels of antigen-specific T cells, memory B cells, and IgG antibodies, but limited mucosal immunity.¹ Conversely, mucosal vaccination more closely mimics the natural infection route, inducing both systemic and mucosal immunity, the latter characterized by mucosal secretory IgA (sIgA) and tissue-resident memory T cell (T_RM) responses.² Additionally, mucosal vaccination provides superior immunity at pathogen entry sites, thus offering the potential to block pathogen transmission and eliminating the need for needles, making it practical for mass vaccination. However, mucosal vaccination presents specific challenges, including the need for immune stimulants like adjuvants to induce a protective immune response efficiently and safely, and the requirement for specialized devices for effective administration due to the compartmentalization of mucosal surfaces.³

A thorough understanding of the immune response mechanisms at mucosal sites is essential for the development of more effective mucosal vaccines. This review first aims to evaluate the characteristics of the respiratory mucosal immune system in the context of mucosal infections and vaccination strategies. It will then discuss recent advancements in mucosal vaccines and adjuvants and highlight the potential of novel adjuvant formulations in addressing critical challenges in the development of respiratory mucosal vaccines.

Anatomy and characteristics of the respiratory mucosal immune system

The respiratory tract is divided into the upper respiratory tract (URT) and lower respiratory tract (LRT). The URT comprises the conducting airways, including the nasal cavity, pharynx, and larynx, which form a continuous passageway for air movement, along with the associated lymphoid tissue, including the nasal-associated lymphoid tissue (NALT), and the cervical lymph nodes, which drain the URT. The LRT contains the trachea, as well as the bronchi and bronchiole branches within the lungs. The anatomical structures of the upper and lower respiratory tracts vary between different age groups and species.⁴ A good understanding of these differences is crucial for the development of safe and effective mucosal vaccines and adjuvant research. Notably, in humans, the nasal cavity is predominantly lined with respiratory epithelium, occupying approximately 90% of the epithelial surface, while the olfactory epithelium constitutes the remaining 10%.⁵ However, in mice and rats, both the respiratory and olfactory epithelia each comprise about 50% of the nasal cavity. Specifically, the upper (larger-diameter) airway in humans primarily consists of ciliated cells and mucus-secreting goblet cells, while the lower (smaller-diameter) airway has fewer goblet cells but more secretory cells.⁶ The mouse trachea has a similar cellular composition as the human bronchiole.

Lymphatic draining from the LRT goes to the bronchial-associated lymphoid tissue (BALT), along with the mediastinal lymph nodes, which together play critical roles in initiating local adaptive immune responses to pathogens infiltrating the lungs (Figure 1). Within these structures of the lung, innate and adaptive immune mechanisms exist to maintain respiratory health and defend against pathogen infiltration.

Figure 1. Organization and cellular characteristics of the human respiratory airway.

PNEC, pulmonary neuroendocrine cell; NALT, nasal-associated lymphoid tissue; BALT, bronchial-associated lymphoid tissue. Figure created with BioRender.com.

The mucosal immune system is classified into inductive sites, which recognize and process antigens (mucosa-associated lymphoid tissue (MALT), including NALT and BALT), and effector sites, which produce sIgA (nasal lamina propria and nasal mucosa).⁵ Additionally, stimulation of specific inductive sites can induce similar immune responses at various effector sites, a critical concept for the design of mucosal vaccine delivery routes.⁷ This unique interconnected circulation of lymphocytes within the mucosal immune system is termed the common mucosal immune system (CMIS). Microfold cells (M cells) within inductive sites capture antigens and facilitate their presentation to dendritic cells (DCs).⁸ These DCs then migrate to the T cell region of the MALT and activate naïve T cells, which in turn stimulate the isotype switch of IgM⁺ B cells to IgA⁺ B cells, a process orchestrated by cytokines including TGF-β and IL-10.⁷ Consequently, IgA⁺ B cells migrate to various mucosal effector sites through the lymphatic system, differentiate into plasma cells, and produce sIgA.⁹ Mucosal tissue-resident IgA-producing plasma cells secrete IgA that polymerizes into polymeric IgA (pIgA) through J-chain linkage.¹⁰ This pIgA, transported across epithelial cells by the polymeric Ig receptor (pIgR), undergoes cleavage to form sIgA, which can prevent microbial attachment to mucosal surfaces by neutralization and aggregation.^7,11 Additionally, the upper and lower respiratory tracts exhibit distinct antibody compositions.^2,11 sIgA predominates in the upper respiratory tract, where it plays a critical role in mucosal immunity by neutralizing pathogens and inhibiting their adherence to epithelial cells.¹² The immune response in the upper respiratory tract is characterized by the mucociliary clearance of sIgA-bound pathogens, effectively preventing colonization. However, both IgA and IgG in the lower respiratory tract are essential for maintaining homeostasis and providing protection against pathogens.^1,13 IgG is critical for opsonization, pathogen neutralization, and activation of complement pathways, contributing to both lower respiratory tract and systemic protection.¹⁴ Furthermore, the defense mechanisms in the lower respiratory tract involve robust IgG-mediated functions, supported by a strong cellular immune response involving alveolar macrophages and T lymphocytes.^1,15

Innate immunity in the respiratory mucosa

Mucosal innate immunity encompasses mechanical, chemical, and cellular elements, which provide first-line defense in the respiratory mucosa. The mechanical elements include a physical barrier of tightly formed epithelium covered with a mucus coating. The ciliated epithelium, along with mucus secreted by goblet cells, expel debris, allergens, and potential pathogens in the URT.¹¹ The same cellular components are also present in the LRT, along with additional presence of club cells, non-ciliated epithelial cells that represent the major secretory cell in the small airway epithelium. These cellular components express various pattern recognition receptors (PRRs) and cytokine receptors, enabling them to continuously survey for pathogens and coordinate immune responses with both local and recruited immune cells.^16–18 Pulmonary endothelial cells (ECs) primarily function as a barrier between the blood and interstitium.¹⁹ ECs also actively participate in immune surveillance, phagocytosis, immune cell recruitment, and antigen presentation, with some classified as immunomodulatory endothelial cells (IMECs) that modulate the balance between immune tolerance and inflammation.

Respiratory mucus consists of two defined layers: an upper viscous, gel-like mucus layer and an underlying periciliary layer (PCL) (Figure 1). These mucus layers are characterized by a bottlebrush-like structure in which mucins, O-glycosylated proteins, are tethered to the airway surface.²⁰ Mucins are the primary components of both layers of mucus and are crucial in safeguarding the respiratory tract against pathogenic infections.²¹ Aside from merely passive blocking of pathogens, dense glycosylation, and sialylation of mucins trap glycan-receptor-binding viruses, aiding in their mucociliary clearance. The mucus layer can play a factor in mucosal vaccine delivery. Retention times of vaccine formulations at mucosal sites are important factors for consideration in mucosal vaccine design, and formulation efforts that can promote mucoadhesion and mucopenetration are intense areas of research.²²

The main innate immune cellular elements include neutrophils, NK cells, macrophages, and dendritic cells.^11,12 These cell types either directly engulf pathogens via phagocytosis or initiate signaling pathways that promote killing upon recognition of conserved structures on the pathogens.¹² Upon infection, neutrophils, being the first responders, are recruited to the lungs and airways.^23,24 Within the respiratory tissue, neutrophils play a critical role in clearing pathogens and cellular debris from the airspaces through degranulation, phagocytosis, and the formation of neutrophil extracellular traps (NETs). NETs are web-like structures composed of cytosolic and granule proteins on decondensed chromatin, which trap and neutralize bacterial cells and sequester excess cytokines, thereby aiding in the regulation of pulmonary inflammation.²⁵

NK cells and macrophages are the frontline innate effector cells that mount the initial response against pathogens.^26–29 NK cells are innate lymphocytes that recognize and kill cancerous or virally infected cells, primarily through secretion of perforin, and granzymes, and mediation of antibody-dependent cellular cytotoxicity, as well as through their significant contribution to the production of interferon-gamma (IFN-γ).^11,30 Macrophages, constituting approximately 70% of the lung’s leukocyte population,³¹ function as phagocytes and sensors of respiratory infections. They express high levels of pattern recognition receptors (PRRs) for detecting infections and have a high propensity for surface Fc receptors, crucial for pathogen clearance. The presence of PRRs makes them good targets for adjuvants, which often are PRR agonists (see below). Vaccine induced non-neutralizing antibodies that engage Fc receptors have been correlated with protection against influenza viruses of different subtypes and provide an extra mechanism for protection beyond virus neutralization.³² Respiratory or airway macrophages can be classified into two groups based on their localization and function: alveolar macrophages (AMs) (long-lived tissue-resident (TR-AMs) and monocyte-derived AMs), and interstitial macrophages (IMs). AMs, the primary phagocytes in the lung, are a distinct subset of self-renewing cells, originating from fetal liver precursors.^11,29,33,34 TR-AMs engulf foreign material and apoptotic cells in the alveolar space and participate in constant crosstalk with epithelial cells within the lung to coordinate immune activation or suppression. TR-AMs provide the first-line defense in the LRT, releasing cytokines and chemokines upon infection to initiate inflammatory responses and recruit other effector cells to the alveolar space. However, AMs can also be exploited by various viruses for dissemination, persistence, and replication. Interstitial macrophages (IMs) are predominantly located between the microvascular endothelium and alveolar epithelium, where they perform phagocytosis and antigen presentation like AMs, and can also modulate tissue repair.^11,29,33,35

DCs are long-lived, and can capture antigens from the airway lumen and migrate to the draining lymph nodes in the NALT and BALT, where they present or cross-present antigens to naïve CD4⁺ and CD8⁺ T cells.¹¹ Dendritic cells are classified into three main groups in the lungs: plasmacytoid DCs (pDCs) and the monocyte-derived conventional subsets, conventional cDC1 and cDC2. pDCs are dispersed throughout the lung tissue, including the airway, lung parenchyma, and lung stroma.³⁶ pDCs are vital in innate immunity due to their substantial production of type I interferons, especially in response to sensing of viral PAMPs, thereby triggering a cascade of antiviral pathways. pDCs typically respond rapidly upon sensing of bacterial or viral nucleic acids at the early stages of infection, and as such, play an important role during the initiation of the host response.³⁷ cDC1s are found in vascular and mucosal areas, initiating Th1 responses and cross-presenting to CD8⁺ T cells to promote cytotoxic functions for the killing of intracellular pathogens. In contrast, cDC2s, located in the lamina propria, prime CD4⁺ T cells and produce pro-inflammatory chemokines, which recruit various inflammatory cells to sites of lesions. Furthermore, cDC2s have been suggested to also help in the establishment of immune tolerance.³⁶ Given their role in antigen presentation, efficient DC activation and antigen delivery to DCs is a crucial step during mucosal vaccination.

Innate immune cells have been increasingly recognized for their ability to develop ‘trained immunity,’ a nonspecific memory-like function.^38–40 Trained immunity is exemplified by a ‘priming’ phase of innate immune cells in response to exposure to specific stimuli, in which epigenetic and metabolic reprogramming is induced in innate immune cells. Trained innate immunity has been ascribed to various innate immune cells such as monocytes, dendritic cells (DCs), natural killer (NK) cells, fibroblasts, and macrophages.^33–42 Since the mucosa of the respiratory tract is continuously exposed to a variety of pathogens, including viruses and bacteria,⁴³ it is clear that trained immunity has an important role in shaping mucosal innate immunity and subsequently downstream adaptive immune responses to both infection and vaccination. The impact of trained immunity on induction of mucosal vaccine responses and the outcome during reinfection is currently not fully understood, but a high priority research area with potential implications for the innate immune response during mucosal vaccination.

Components of the mucosal adaptive respiratory immune response

The respiratory adaptive immune system involves several key players. This includes CD8⁺ cytotoxic T lymphocytes (CTLs), which kill infected cells upon recognition of class I MHC-bound peptides.^44,45 CTLs exert such activity through the secretion of cytokines, the release of cytotoxic granules, and through the induction of apoptotic pathways in target cells. CTLs are critical for eliminating viral and intracellular bacterial infections in the lung as well as in eliminating tumor cells; however, they can also cause collateral lung damage if dysregulated. Another key player is the CD4⁺ T cells, traditionally known as ‘helper’ cells, which enhance cytotoxic responses or stimulate B cell antibody production.¹² While multiple less abundant CD4⁺ T cell subsets have been recently defined, the five major CD4⁺ T cell subsets include Th1, Th2, Th17, regulatory T (T_reg), and T follicular helper (T_FH) cells. These subsets are classified based on the signature cytokines they produce and their lineage-specific transcription factors. Th1 cells, which respond to intracellular lung infections through enhancing both innate and adaptive cytotoxic responses, produce IFN-γ, IL-2, and TNF-$\mathit{\alpha }$ and express T-bet. Th2 cells regulate airway inflammation and influence B-cell differentiation, antibody production, and eosinophil recruitment through secretion of IL-4, IL-5, and IL-13, and express GATA-3. The Th17 subset secretes IL-17A, IL-17F, IL-21 and IL-22, and express RoRγt. Th17 cells are especially important to host defense and homeostasis at mucosal sites, and their secreted cytokines lead to the recruitment of neutrophils as well as B cells to sites of infection, and can enhance the efficiency of APCs and the CTL response.⁴⁶ T_FH cells, which produce IL-21 and express Bcl6, are essential to driving antigen-specific antibody responses through their function in germinal centers (GCs) formation and B cell maturation within these GCs. Finally, T_reg express FoxP3 and CD25 and act to regulate immune responses and mediate airway tolerance and tissue repair to avoid immunopathology arising from overexuberant inflammatory responses.⁴⁵

Following an infection or vaccination, long-lived adaptive responses form through the establishment of memory T and B cells.¹¹ Of particular interest is the differentiation of subsets of T and B lymphocytes in the mucosae into tissue-resident memory (RM) lymphocytes, which are non-circulating and long-lived.⁴⁷ Unlike their circulating counterparts, these lymphocytes are specifically adapted to provide rapid and localized immune responses to repeated pathogen exposure. In the context of respiratory infections and mucosal vaccination, CD8⁺ T_RM are particularly significant. These cells are identified by their expression of CD69, CD103, CXCR3 and reduced expression of the chemokine receptors, CCR7 and CD62L, enabling them to remain within peripheral tissues.⁴⁸ Functionally, CD8⁺ T_RM aid in rapid viral clearance through the production of various cytokines (IFN-γ, TNFα, IL-2) and cytolytic molecules like granzyme B.⁴⁹ CD4⁺ T_RM cells are more abundant than CD8⁺ T_RM and play a crucial role in pulmonary defense.⁵⁰ These cells, often found around airways and within special lymphoid structures in the lungs, help in the development of CD8⁺ T_RM and support B cells in antibody production. After lung infections, CD4⁺ T_RM cells localize around the airways and within inducible bronchus-associated lymphoid tissues (iBALT), forming clusters with other immune cells such as follicular dendritic cells and other APCs.⁵¹ iBALT, a tertiary lymphoid organ distinct from BALT, is formed in response to antigenic stimuli including infections, self-antigens, and other sources.⁵² Similarly, tissue-resident memory B cells (B_RM) are persistent cells located in mucosal tissues after infection that can quickly respond upon re-encountering antigen.⁵³ Notably, the localization of B_RM varies among species. For instance, in the upper respiratory tract, B_RM cell is situated in the tonsils and adenoids in humans, and in NALT in mice.⁵ In the lower respiratory tract, B_RM cells are found within iBALT or dispersed near alveoli, independently of iBALT.⁵⁴ Unlike circulating B cells, lung B_RM engage in tissue scanning and transform into plasma cells upon encountering an antigen, enhancing local antibody production for quicker pathogen elimination. Studies involving the transfer of these cells have demonstrated that lung B_RM effectively reduce viral levels in the lower respiratory tract, more so than memory B cells from the spleen.⁵⁵

Mucosal vaccination

Intramuscular vaccination often results in a suboptimal mucosal immune response, leading to the proposition that protective mucosal immunity may require mucosal vaccination routes.⁵⁶ Mucosal immunity would not only prevent establishment of respiratory infection but may also reduce transmission to new hosts by reducing virus shedding from mucosal sites.⁵⁷ Nasal and pulmonary delivery of vaccines is particularly effective in eliciting both a local and a systemic immune response. Mucosal immunization generates antigen-specific IgA antibodies at the site of vaccine administration and systemic IgG antibodies, and stimulates cell-mediated responses as well, including helper CD4⁺ T cells and CD8⁺ cytotoxic T lymphocytes.⁴ Furthermore, mucosal vaccination can efficiently induce long-lasting B- and T-cell memory.¹⁴ However, in mucosal vaccine design, the route of administration and vaccine formulation affecting immunogenicity must be considered.^18,58

As mentioned before, localized immunization at mucosal induction sites tends to enhance antigen-specific IgA responses in effector sites that are immunologically and physiologically adjacent or related. For example, nasal, oral, or intratracheal immunization preferentially boosts specific IgA levels in the corresponding mucosal tissues. Therefore, targeted vaccine delivery through specific mucosal routes may optimize antigen-specific immunity in the desired location (Table 1).

Table 2. Licensed mucosal vaccines.

Pathogen	Trade name (Manufacturer)	Administration /formulation	Format	Mechanism of protection
Influenza	FluMist Quadrivalent* (MedImmune)	Intranasal/Spray	Live attenuated (cold-adapted) vaccine (contains four vaccine virus strains: an A/H1N1 strain, an A/H3N2 strain and two B strains)	Haemagglutinin- and neuraminidase-specific mucosal IgA and systemic IgG responses; possibly a role for cell-mediated immunity; heterotypic protection effective in children
Rotavirus	Rotarix*,** (GlaxoSmithKline Biologicals)	Oral/Liquid	Live attenuated rotavirus vaccine (derived from the human 89–12 strain which belongs to G1P type)	Mucosal IgA and systemic neutralizing IgG antibodies specific for homotypic or heterotypic VP4 and VP7 antigens
	RotaTeq*,** (Merck Sharp & Dohme)	Oral/Liquid	Live attenuated rotaviruses.
Cholera	Vaxchora* (Emergent Travel Health)	Oral/Liquid	Live, attenuated 01 serogroup – ctxA attenuation	Gut antitoxin- and CTB-specific IgA and antibacterial and LPS-specific antibodies
	Euvichol** (EuBiologics)	Oral/Liquid	Heat and formaldehyde-inactivated O1 serogroups + 0139	Mucosal IgA in gut and systemic neutralizing IgG antibodies specific for inactivated bacteria
	Shanchol** (Shantha Biotechnics)	Oral/Liquid	Heat and formaldehyde-inactivated O1 serogroups + 0139
	Dukoral** (Valneva)	Oral/Liquid	Heat and formaldehyde-inactivated O1 serogroups + CTB
Poliovirus	Biopolio (bOPV)** (Bharat biotech)	Oral/Liquid	Live attenuated poliomyelitis type 1 and type 3 viruses – Sabin strain	–
	mOPV and tOPV	Oral/Liquid	Culture passage attenuated polioviruses 1, 2 and 3 serotypes
Typhoid	Ty21a* (Berna Biotech)	Oral/Liquid	A live attenuated vaccine for oral administration only. The vaccine contains the attenuated strain Salmonella typhi Ty21a (1,2).	Mucosal IgA and systemic IgG antibody responses and CTL responses
Recurrent urinary tract infections	UROMUNE*** (Inmunotek)	Oral (sublingual)/Spray	Whole cell-inactivated	Mucosal IgA in urinary tract and systemic neutralizing IgG antibodies specific for inactivated bacteria

*Approved for Use in the United States by Food and drug administration.

**Prequalified or qualified by WHO.

***Approved for use in Canada.

CTB, cholera toxin B subunit; mOPV2, monovalent type 2 oral polio vaccine; tOPV, Trivalent oral polio vaccine.

The intrinsic properties of each mucosal site must also be considered when assessing the proper route of vaccine administration. Oral vaccination, in which the vaccine is swallowed, provides easy access and less discomfort which allows large-scale vaccine rollouts and higher compliance, and also has a better safety profile compared to intranasal administration as it avoids the potential risk of antigen migration to the brain through olfactory epithelium and bulbs.⁵⁹ However, as the harsher environment of the GI tract and the presence of specialized oral tolerance mechanisms remain major challenges for this route, and sublingual or buccal vaccination, which deliver antigen into non-keratinized epithelium and thinner cell layers, may be more promising. Sublingual/buccal vaccination can generate mucosal immune responses in the respiratory, digestive, and reproductive tracts, whereas immunity induced by oral immunization has been mostly restricted to the digestive tract.⁶⁰ Intranasal vaccine administration presents several advantages such as the lack of acidity, and secreted vaccine-degrading enzymes.⁶⁰ When rationally designing a mucosal vaccine formulation, one has to keep in mind that factors such as particle size, surface charge, and hydrophobicity affect vaccine immunogenicity. Particle size between 1–5 µm are considered ideal for lung deposition.^61,62 Furthermore, distinct particle sizes are favored for uptake by different cell types within the lung and draining lymph nodes. Larger particles are typically taken up by DCs at injection site and transported to the draining lymph nodes, whereas smaller particles may drain directly to the lymph nodes before being taken up by lymph node-resident DCs and macrophages.⁶³ Surface charge also affects the cellular uptake of particles.⁶⁴ Fromen et al. suggested that cationic nanoparticles were mostly associated with DCs, whereas anionic particles were mostly internalized by alveolar macrophages.⁶⁴ Additionally, hydrophobic particles easily associate within the lipid bilayer of cells, promoting uptake for further immune processing. In contrast, hydrophilic polymers such as poly ethylene glycol, and poly carboxybetaine were shown to be efficient in decreasing production of TNF-$\text{alphaa}$ and IL6 in BALF of mice.⁶⁵ Although, particle size, surface charge, and hydrophobicity of vaccines are known to influence immune responses in parenteral administration routes, the specific role they play in mucosal vaccines is yet to be fully explored.

Current status of mucosal vaccines

Over the past several decades, there has been a significant shift in traditional injectable vaccines – from inactivated or attenuated vaccines – toward adjuvanted subunit, viral vectored, DNA, and mRNA vaccines.⁶⁶ Many of these newer generation vaccines are currently tested for their feasibility as mucosal vaccines. However, several obstacles exist in the field of mucosal vaccination, including tolerance toward orally administered inactivated antigens, susceptibility of unprotected subunit antigens to degradation and clearance, as well as the absence of established mucosal adjuvants to overcome these issues.^12,13 Multiple vector and formulation methods are currently being tested to optimize mucosal vaccine delivery and effectiveness.^67,68 Given the limited efficacy associated with mucosal immunization methods, few vaccines have been licensed for mucosal administration. Currently, licensed mucosal vaccines are limited to gastrointestinal diseases or and mucosal vaccines that target respiratory mucosae are only available for influenza. They typically consist of the inactivated whole or live attenuated pathogens (Table 2). FluMist, which was approved in 2003, and consists of trivalent cold-adapted influenza viruses is an intranasally administered live attenuated vaccine.⁵ The vaccine has been approved for use in non-immunocompromised individuals aged 2–49. Treanor et al. showed that FluMist-immunized subjects demonstrated a slightly higher protective efficacy against wild-type influenza virus infection compared to the trivalent inactivated influenza virus vaccine (TIV) in a human study. In the studies, 45% (14/31) of the placebo group showed laboratory-confirmed influenza following exposure to the wild-type influenza virus, compared to 13% (4/32) of those receiving the TIV group and 7% (2/29) of FluMist group.⁶⁹ Interestingly, in this study FluMist was more effective in preventing respiratory illness upon experimental challenge than TIV despite the low antibody response rates observed in serum.

Table 1. Expression of mucosal IgA immune responses after different routes of vaccination.

	Route of Administration
	Oral^Citation57	Sublingual/buccal^Citation16,Citation57	Nasal^Citation16	Ocular^Citation16	Vaginal^Citation16	Rectal^Citation16
Upper respiratory tract	–	+++	+++	++	–	–
Lower respiratory tract	–	+++	+++	N/A	–	–
Stomach	+/+++	+++	–	N/A	–	–
Small intestine	+++	+++	–	N/A	–	–
Colon	+	N/A	–	N/A	–	–
Rectum	+	N/A	–	N/A	–	++
Genital tract	–	+++	++	++	++	–
Blood	+	++	+++	-/+	-/+	-/+

Current licensed vaccine adjuvants

Adjuvants are critical components to enhance the uptake and/or immunogenicity of antigens.^70–72 Adjuvants primarily enhance immunogenicity by stimulating antigen-presenting cells (APCs) to enhance antigen presentation and co-stimulatory signals.⁷¹ Despite numerous attempts to develop novel adjuvants, only a limited number have been licensed (all for parenteral administration), such as aluminum adjuvants,⁷³ MF59,⁷⁴ AS01,⁷⁵ AS03,⁷⁶ AS04,⁷⁷ CpG 1018,⁷⁸ and Matrix-M⁷⁹ (Figure 2). Since these adjuvants have been reviewed in depth elsewhere, we will only focus on them when relevant for mucosal vaccination strategies.

Figure 2. Simplified diagram of the major signaling pathways of classical adjuvants.

NLRP3, NOD-, LRR- and pyrin domain-containing protein 3; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain. Figure created with BioRender.com.

Mucosal vaccine adjuvants

No adjuvants specifically for nasal vaccinations have been licensed for human use.⁸⁰ Antigen-only mucosal vaccinations often fail to elicit a robust immune response that provides enduring protection against infections.¹⁵ One strategy for developing adjuvants for mucosal vaccines is to employ the above-mentioned adjuvants that have been approved for parenteral human vaccine use.^13,80–82 Aluminum salt, the first adjuvant to receive licensing, has undergone extensive study in various nasal vaccination formulations, in both dry and liquid vaccines.⁸¹ Thakkar et al. demonstrated the efficacy of an intranasal dry powder vaccine comprising ovalbumin and aluminum salt adjuvants.⁸³ The intranasal immunization in rats with this dry powder vaccine with aluminum salt adjuvant induced specific antibody responses in both serum and mucosal secretions. Furthermore, a recent study proposed using porous aluminum-based metal-organic framework nanoparticles (MOF NPs) as inhalable adjuvants.⁸¹ When compared to traditional alum, these MOF NPs had superior capacity to activate APCs, stimulate higher IgG2a titer, and elevate Th1 responses, when assessed using ovalbumin as a model antigen.⁸¹

AS04 has also shown potential in enhancing mucosal and systemic immunity, when utilized in intranasal vaccines.⁸² Intranasal administration of AS04-adjuvanted vaccines in mice elicited both mucosal (IgA in nasal and lung mucosae) and systemic immune (IgG in the serum) responses, in contrast to the solely systemic response observed with subcutaneous administration. AS04 demonstrated superior antigen deposition in the nasal cavity in mouse models.⁸⁴

An intranasal H5N1 influenza vaccine containing a virosomal formulation adjuvanted with Matrix-M exhibited substantial protection in mice against viral challenge with 100 LD₅₀.⁸⁴ This vaccine stimulated a well-balanced immune response, generating both Th1 and Th2 cytokines.

As an alternative to repurposing parenteral vaccine adjuvants for mucosal administration, there is a focus on pioneering novel mucosal adjuvants, including those based on pattern recognition receptor (PRR) agonists, toxoids, and muco-adhesive systems.

Agonists of PRRs

Mechanistically, many adjuvants activate PRRs on innate immune cells, including Toll-like receptors (TLRs), STING agonists, C-type lectin receptors (CLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), and NOD-like receptors (NLRs). Activation of these receptors triggers signaling cascades that lead to dendritic cell maturation, cytokine secretion, and T helper cell stimulation.⁸⁰ TLR agonists, widely recognized as potential adjuvants for parenteral vaccines, have also been extensively researched as key mucosal adjuvants, including Pam3CsK4 (TLR2), Polyinosinic-polycytidylic acid (Poly I:C, TLR3), Monophosphoryl Lipid A (MPL, TLR4), flagellin (TLR5), gardiquimod (TLR7/8), and CpG-oligodeoxynucleotide (CpG-ODN, TLR9).⁸⁰

Pam3CSK4, a TLR2 agonist, has demonstrated efficacy in intranasal RSV virosomes vaccines, inducing serum IgG, mucosal antibodies and safe Th1-skewed cellular immune responses, without priming for enhanced disease in mice.⁸⁵ Additionally, utilization of synthetic macrophage-activating lipopeptide-2 (MALP-2), a TLR2/6 agonist, as an adjuvant has demonstrated enhancement of systemic and mucosal antibody responses, as well as robust cell-mediated immunity in animal models.⁸⁶

Poly I:C, a synthetic double-stranded RNA analogue that activates TLR3, has been studied extensively as a nasal vaccine adjuvant. Sabbaghi et al. explored the immunological effects of intranasal immunization in mice using a gamma-irradiated H1N1 vaccine augmented with Poly I:C and/or chemokine (C–C motif) ligand 21 (CCL21).⁸⁷ This was evidenced by an increase in the ratio of IgG2a, IFN-γ, and IL-12, which are markers of Th1 immune responses. Additionally, there was a notable elevation in both IgA- and IgG-mediated humoral immunity. The study also observed increased lymphocytic proliferation and enhanced cytotoxic activity in spleen cells. The potential of flagellin (a TLR5 agonist)^88,89 and CpG-ODN TLR9 agonist)^90,91 in mucosal adjuvant applications has been investigated in various studies. These investigations have revealed that both flagellin and CpG-ODN demonstrate significant promise as mucosal vaccine adjuvants in preclinical studies.

Among the PRRs, RIG-I agonists have been proposed as novel adjuvants for mucosal vaccines.^92,93 RIG-I receptors are in the cytosol and recognize short double-stranded RNA (dsRNA), a replication intermediate for RNA viruses, that exhibit viral motifs such as an uncapped 5′ diphosphate or triphosphate end. We have innovatively developed an intranasal adjuvant, combining an RNA-based RIG-I agonist (IVT DI) with a nanoemulsion (NE) that activates TLRs and NLRP3, to enhance immune responses.^94–96 The NE formulation of this intranasal adjuvant can also serve as a delivery vehicle for IVT DI and antigen as well, enhancing the retention time of the nasal vaccine at mucosal sites and improving cellular uptake of the antigen and agonist cargo. The NE/IVT adjuvant, when paired with the recombinant SARS-CoV-2 receptor binding domain (RBD) protein, has been shown to induce strong and lasting humoral, mucosal, and cellular immunity.⁹⁴ Furthermore, NE/IVT with the ancestral SARS-CoV-2 spike protein antigen elicited enhanced Th1-biased cellular responses and high titers of virus-neutralizing antibodies effective against a broader range of divergent SARS-CoV-2 variants, including those of the Omicron lineage.⁹⁵ Notably, it elicits the production of antigen-specific IFN-γ/IL-2/TNF-α, which is crucial for protective immunity, especially in the elderly.⁹⁴ In contrast to the licensed MF59-like adjuvant, Addavax, our NE/IVT formulation maintains its immunogenic effectiveness in preclinical experiments, regardless of age. Our studies and those of others suggest that optimal mucosal adjuvant development will likely benefit from the integration of multiple classes of adjuvants and delivery systems.

Double-mutant heat-labile toxin and multiple mutated cholera toxin

E. coli heat-labile toxin and Cholera toxin are considered among the top mucosal adjuvants.⁹⁷ However, due to their toxicity, there has been a push to improve safety while maintaining their adjuvant properties. This effort has led to the creation of modified versions such as the E. coli double-mutant heat-labile toxin (dmLT) and the multiple-mutated cholera toxin (mmCT). The inclusion of dmLT has notably enhanced responses to whole-cell antigens in vaccines like ETVAX, which is an oral, inactivated, enterotoxigenic E. coli vaccine.⁹⁷ Novel adjuvants including mmCT show potential in boosting Th1 and Th17 cellular responses.⁹⁸ CTA1-DD, a detoxified derivative of cholera toxin, has been effective in stimulating immune responses in (neonatal) mice and shows promise in influenza vaccination, although its clinical efficacy and safety are yet to be fully established.⁹⁹ However, the use of some toxoid adjuvants in nasal delivery, the most well-documented type of mucosal adjuvants, has been linked to adverse effects like Bell’s palsy, leading to the exploration of alternative methods like sublingual administration.¹⁰⁰ Recent clinical trials with another genetically detoxified heat-labile enterotoxin (LT)-derived from E. coli (LTh(αK)), have shown promising results in nasal vaccination without adverse effects.^101,102 Overall, toxoid adjuvants represent a significant advancement in mucosal adjuvant technology, particularly for oral vaccine formulations with whole-cell killed pathogens.

Chitosan

Finally we want to mention chitosan as an example for natural polysaccharides, which have been increasingly recognized for their roles as mucosal adjuvants due to their capacity to activate a range of immune cells and augment the secretion of cytokines such as, IL-4 and IFN-γ as well as their muco-adhesive properties.¹⁰³ Chitosan has garnered interest in the field of mucosal adjuvants owing to its low toxicity, excellent biocompatibility, and biodegradability.¹⁰⁴ Additionally, chitosan has unique cationic properties, allowing it to adhere well to the mucosal surface, which not only prolongs the retention time of antigens but also opens tight junctions between mucosal epithelial cells so antigens can be more readily accessed by mucosal APCs.¹⁰⁵ A variety of chitosan derivatives have been explored as innovative mucosal adjuvants for different vaccine types.^106–108

Conclusions

Despite the many unique challenges, mucosal vaccination strategies have much potential in controlling respiratory pathogens. Mucosal vaccination strategies have potential in preventing both disease and transmission of pathogens, two crucial factors during infectious disease outbreaks with epidemic or pandemic potential. Therefore, there is great interest in mucosal vaccine development. We have highlighted some of the hurdles that need to be addressed during that process. It is likely that better adjuvant candidates, as well as novel formulation methods and vaccine vectors for mucosal vaccination than those currently licensed for parenteral administration, will move forward to the clinic in the coming years. Safety will remain a main driver of mucosal vaccine research, alongside effectiveness. A better understanding of the mucosal immune system will allow for the development of more targeted strategies. Here, we mainly focused on the mucosal sites of the respiratory tract, which comprises only part of the mucosal immune system. Interestingly, it became clear in recent years that significant immune cross talk between distant mucosal sites exists.¹⁰⁹ Therefore, mucosal vaccination at inductive sites that are easily accessed (nasal, oral) can also be targeted to induce mucosal immunity against pathogens that, for example, colonize the gut or vaginal mucosae. Novel vaccine technologies that focus on mucosal vaccine delivery, stability, and immunogenicity along with our rapidly expanding understanding of the mucosal immune system and the mucosal immune correlates of protection gained through more powerful systems immunological tools will together enable development of the next generation of mucosal vaccines.

Acknowledgments

Research on mucosal immunology and vaccination in the Schotsaert and Wong laboratories is supported by NIAID grants R21AI176069 and R01AI160706. S.C.P. has received support from the Korea Health Industry Development Institute (KHIDI) for research under the Biomedical Global Talent Nurturing Program (HI22C2101).

Disclosure statement

The M.S. laboratory received unrelated research support as sponsored research agreements from ArgenX BV, Phio Pharmaceuticals, 7Hills Pharma LLC and Moderna. The remaining authors declare no conflict of interest.

Additional information

Funding

The work was supported by the Korea Health Industry Development Institute [HI22C2101]; National Institutes of Health [R01AI160706, R21AI176069].