Recent advances in respiratory immunization: A focus on COVID-19 vaccines

Abstract

The development of vaccines has always been an essential task worldwide since vaccines are regarded as powerful weapons in protecting the global population. Although the vast majority of currently authorized human vaccinations are administered intramuscularly or subcutaneously, exploring novel routes of immunization has been a prominent area of study in recent years. This is particularly relevant in the face of pandemic diseases, such as COVID-19, where respiratory immunization offers distinct advantages, such as inducing systemic and mucosal responses to prevent viral infections in both the upper and lower respiratory tracts and also leading to higher patient compliance. However, the development of respiratory vaccines confronts challenges due to the physiological barriers of the respiratory tract, with most of these vaccines still in the research and development stage. In this review, we detail the structure of the respiratory tract and the mechanisms of mucosal immunity, as well as the obstacles to respiratory vaccination. We also examine the considerations necessary in constructing a COVID-19 respiratory vaccine, including the dosage form of the vaccines, potential excipients and mucosal adjuvants, and delivery systems and devices for respiratory vaccines. Finally, we present a comprehensive overview of the COVID-19 respiratory vaccines currently under clinical investigation. We hope this review can provide valuable insights and inspiration for the future development of respiratory vaccinations.

Graphical Abstract

1. Introduction

The newly recognized contagious respiratory disease, known as Coronavirus Disease 2019 (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become as widespread as a pandemic. As of December 2022, over 620 million confirmed cases of COVID-19 have been recorded globally [1], and the outbreak is projected to last until 2025 [2]. Due to the rapid spread of SARS-CoV-2, the development of an effective vaccine as one of the most cost-effective ways to combat COVID-19 is essential to protecting the global population. As of November 1, 2022, there are 371 vaccine candidates against SARS-CoV-2 worldwide, of which 199 are in preclinical development and 172 are in clinical development [3]. The route of vaccination, including intramuscular, subcutaneous, intradermal, oral, and intranasal administration, can impact vaccine efficacy and safety as well as the type of immune response elicited. For example, compared to subcutaneous injections that deliver antigens into the fatty layer of tissue between the skin and muscle, intramuscular injections deliver antigens directly into the more highly vascularized muscle mass, allowing for more rapid absorption and processing of vaccine components [4]. In addition, intramuscular injections could also reduce local adverse effects associated with adjuvants. Therefore, intramuscular injection is the most commonly used method of vaccination to date [5]. Statistically, the World Health Organization (WHO) has granted emergency use listings (EUL) to 11 vaccines, all of which are delivered intramuscularly, and 78% of the clinically developed SARS-CoV-2 vaccines are administered intramuscularly as well [3]. However, most of these intramuscular COVID-19 vaccines only prevent severe disease and cannot efficiently eliminate disease transmission, as they do not provide sterilizing immunity [6]. Actually, many vaccinees could still become asymptomatic carriers [7]. Sterilizing immunity, which is the ideal goal for all vaccines against infectious diseases, provides protection through neutralizing antibodies that inhibit infection and multiplication of the virus in the vaccinated individual, ultimately preventing transmission [[7], [8], [9]]. For example, human papillomavirus (HPV) subunit vaccines provide sterilizing immunity through eliciting high-titer, durable antibody responses in the genital tract [10]. Mechanism studies showed that intramuscular administration of this virus-like particle-based subunit vaccine induced HPV-specific IgA and IgG responses. These antibodies are present not only in the serum but also transport from the plasma into the mucosal barrier via exudation at breaches in the cervicovaginal epithelium or via active transcytosis [11,12]. However, the intramuscular COVID-19 vaccine only elicits strong IgG responses and not mucosal secretory IgA (sIgA) responses, while circulating IgG cannot effectively protect the upper respiratory tract from infection [6,13,14]. Therefore, mucosal vaccines that can stimulate sIgA production on the mucosal surface of the respiratory tract and that can thereby provide sterilizing immunity by blocking the virus at the entry point and preventing the initial replication of the virus, would be preferable [15,16]. Additionally, the intramuscular injection has low compliance due to needle phobia, logistical limitations of cold-chain transport, and the necessity for trained medical professionals, which dramatically diminishes the possibility of rapid mass vaccination, particularly in developing nations. As a result, researchers are focusing on improving vaccine formulations and the routes of vaccine administration, making respiratory vaccines a viable alternative for the prevention of COVID-19 infections.

Respiratory administration of the COVID-19 vaccine, via the nose and lungs, represents a highly promising mode of immunization. First, SARS-CoV-2 is an infective mucosal pathogen that can be transmitted between individuals by respiratory droplets [17]. The virus utilizes its spike (S) receptor-binding domain (RBD) to bind to angiotensin-converting enzyme 2 (ACE2) receptors, which are expressed in type II alveolar, goblet, and ciliated cells in the airways [[18], [19], [20]]. This allows SARS-CoV-2 to infect the human respiratory tract [21]. Additionally, ACE2 expression is present on cells in many other organs, including the liver, gut, kidney, and heart [20,22,23], contributing to their susceptibility to SARS-CoV-2 infection. Notably, the respiratory COVID-19 vaccine delivers antigens directly to the entry ports of the pathogen [24], the nose and the pulmonary, and therefore can generate durable systemic immune responses and mucosal immunity (sIgA) in the upper and lower respiratory tracts [17,[25], [26], [27]], effectively preventing SARS-CoV-2 infection, replication, shedding, and transmission [17]. Second, the administration of respiratory vaccines offers distinct advantages in terms of safety and convenience. Non-invasive delivery eliminates the need for medical professional administration, reducing needle phobia, and increasing patient compliance. In addition, the respiratory route of delivery eliminates the risk of transmitting pathogens such as the Human Immunodeficiency Virus (HIV) and Hepatitis B virus (HBV) associated with improper needle use [5,24]. Moreover, the utilization of simple administration devices obviates the need for a sterile environment, making vaccination efforts more feasible in resource-limited regions. Furthermore, mucosal immunization of the respiratory tract presents a new strategy for vaccination by incorporating intramuscular and respiratory immunization methods. Intramuscular prime and intranasal booster vaccinations have the potential to induce better well-rounded immune responses and provide more effective infection prevention compared to parenteral vaccines [17]. Also, the “prime-boost” strategy involves utilizing homologous or heterologous vaccination, which refers to the administration of the same or different vaccine platforms [28]. Recently, several investigations have indicated that heterologous prime-boost immunization strategies could potentially enhance the efficacy of the COVID-19 vaccine [[29], [30], [31], [32], [33]]. For instance, Iwasaki et al. proposed a “prime and spike” approach that combined these two immunization strategies [34]. The "prime" aspect refers to the generation of existing immunity through the intramuscular injection of the mRNA-LNP vaccine (Comirnaty), followed by the induction of mucosal immunity in the respiratory tract using different spike proteins of coronavirus as heterologous boosters (spike). Their results demonstrated that the “Prime and Spike” approach elicited robust IgA, B and T resident memory cells in the respiratory mucosa and induced strong cross-reactive immunity against sarbecoviruses in comparison to parenteral vaccines alone. Consequently, the development of respiratory vaccines is highly promising, and numerous COVID-19 respiratory vaccines are currently under development globally, with ten of these vaccines in the clinical stages [3]. Table 1 presents a comparison of the characteristics, advantages, and limitations of intramuscular and respiratory vaccines. In this review, we describe the structure of the respiratory tract and the mechanisms of respiratory mucosal immunity, discuss in detail the potential adjuvants, delivery systems, and devices for use in respiratory vaccines, provide insights into the development of respiratory COVID-19 vaccine formulations, and conclude with an updated and comprehensive summary of the respiratory COVID-19 vaccines currently in clinical investigation.

Table 1.

Comparison of intramuscular and respiratory vaccines

	Intramuscular	Respiratory tract
Administration method	Hypodermic Needle	Delivery device (Nasal spray, Nebulizer, Inhaler)
Immune response	Systemic humoral immune response (IgG) and T cell responses	Systemic (IgG) and mucosal (sIgA) humoral immunity, and T cell responses
Efficiency of protection	Protects the lower respiratory tract but does not prevent the spread of viruses	Limit the viral transmission
Adjuvant	Adjuvants for human use are available	Absence of safe and effective mucosal adjuvants for human
Self-administration	No (Requires trained medical professionals)	Yes
Pain	Painful (Needle stick injuries)	Painless (Noninvasive)
Patient compliance	Low	High
Risk of delivery	Risk of pathogen transmission associated with improper needle use	Low risk
Transportation and storage	Cold chain	No requirement for cold chain
Mass vaccination	Limited	Suitable
Authorized human use	Most vaccines	Only two nasal vaccines

2. The immune system of the respiratory tract

2.1. The structure of the respiratory tract

The respiratory system is anatomically comprised of the upper respiratory tract (URT) and the lower respiratory tract (LRT). Generally, the upper respiratory tract encompasses the nasal cavity, pharynx, and part of the larynx above the vocal folds and is regarded as the primary site of infection for the majority of pathogens, including SARS-CoV-2. The lower respiratory tract includes the trachea, bronchi, and lungs [[35], [36], [37]]. Functionally, the respiratory tract can be viewed as comprising two components: the conduction component and the gas exchange component [38,39] (Fig. 1). The conduction portion, commonly referred to as the conducting airway, extends from the nasal cavity to the terminal bronchioles [38] and is responsible for conducting, warming, moistening, and cleaning the air [40]. Located within the lung parenchyma, the gas exchange portion transports gases (especially carbon dioxide and oxygen) in and out of the capillaries in the lungs [36,40].

Fig. 1.

Schematic diagram of the conduction and gas exchange portion. In the respiratory tract, conducting airways continued until the 16th generation. The airways in the lower part of the respiratory system (16th to 23rd generations) are responsible for the exchange of gases and play an essential role in immune defense. The figure was reproduced with permission from ref. [41]. Copyright 2022 Elsevier.

In addition, the respiratory epithelium consists of tightly connected epithelial cells on the surface of the lumen, including ciliated columnar cells, brush cells, basal cells, goblet cells, and small granular cells [42]. These cells serve as an essential mechanical-physical barrier against pathogens or hazardous substances that are inhaled [43]. The epithelium of the respiratory tract, along with the lamina propria, makes up the respiratory mucosa, which secretes a thick layer of protective mucus [38]. The mucosal immune system of the respiratory tract, including both the innate and adaptive immune systems, acts as a vital barrier against harmful pathogens [41].

2.2. The mucosal immune system of the respiratory tract

The mucosal immune system can be classified into two components based on anatomical localization and function: the inductive site and the effector site [44,45]. The inductive site consists of regional mucosa-draining lymph nodes and organized mucosa-associated lymphoid tissues (MALTs) [46,47], which recognize and uptake antigens and initiate antigen-specific immune responses [48]. Bronchus-associated lymphoid tissue (BALT) and nose-associated lymphoid tissue (NALT) in the respiratory system are parts of MALT [49,50]. The NALT of rodents is a pair of aggregated lymphoid tissues positioned near the nasopharyngeal duct entrance on the dorsal side of the soft palate [[50], [51], [52], [53]]. In humans, the NALT consists of the tubal, palatine, lingual, and pharyngeal tonsils (adenoids) [53,54], which corresponds to the Waldeyer’s ring [55]. The BALT is commonly located at the bifurcation of the bronchi and bronchioles, and consists of lymphoid follicles in the lungs and bronchus [56]. Variations in BALT exist among species, with presence in rabbits and rats but not typically in healthy mice or adult humans [57]. Human lungs would develop tertiary lymphoid organs known as inducible BALT (iBALT) when exposed to high antigenic loads [57,58]. Effector sites, such as the secretory glandular tissues and lamina propria regions, contain antigen-specific mucosal effector cells [47,59], including IgA-producing plasma cells and B- and T-lymphocytes [60].

When pathogens enter the respiratory tract, the first barrier against viral invasion is the respiratory epithelial layer [61]. Apart from the well-known physical barrier established by tightly connected epithelial cells, the mucociliary clearance system also plays a crucial role in protecting respiratory tract tissues. This system mainly consists of the cilia found on columnar ciliated cells as well as the mucus secreted by goblet cells and submucosal glands [62,63]. Ciliated cells are found in abundance in the respiratory epithelium, and as the size of the airways in the lungs decreases, the proportion of ciliated cells decreases with increasing cilia height [64]. The mucus of the respiratory tract is a viscoelastic gel made up of mucins (high molecular weight glycoproteins) [65], water, ions, proteins, and lipids [66]. They form a dense protective mucosal blanket that traps inhaled pathogens and particulates [35]. Simultaneously, the trapped material is swept from the lungs to the pharynx by the coordinated beating of the cilia for final clearance [62,67].

2.2.1. Innate immune response

The innate immune system provides the other line of defense against harmful pathogens. A variety of immune cells in the upper airway mucosa play key roles in innate immunity, including macrophages, natural killer (NK) cells, phagocytic neutrophils, and mast cells [68]. These immune cells provide the innate immunity in multiple ways, such as phagocytosis, cytotoxic means, the recruitment of inflammatory cells, etc. [69,70]. In addition, defensive compounds present in mucus, such as defensins, lactoferrin, lysozyme, cathelicidin LL37, and PLUNC (palate, lung and nasal epithelium clone) proteins, also contribute to innate immune defense [[71], [72], [73]]. For instance, several studies have revealed that lactoferrin exhibits antiviral properties against RNA and DNA viruses by inhibiting the binding of viruses to host cells [74,75]. Another study found that lactoferrin can keep host cells from being infected by SARS-CoV [76] and also has anti-inflammatory and immunoregulatory effects [77].

When pathogens reach the deeper airways or lungs, they come into contact with the alveolar lining fluid, which contains surfactants [78,79]. Pulmonary surfactants are endogenous lipoprotein complexes produced by type II alveolar cells and primarily composed of phospholipids (particularly dipalmitoylphosphatidylcholine) and four surfactant-associated proteins (SP-A, SP-B, SP-C, and SP-D) [80]. Notably, SP-A and SP-D, which are soluble collectins, have been shown to protect epithelial cells against infection through viral neutralization, agglutination, and enhanced phagocytosis [81]. Thus, pulmonary surfactants are believed to opsonize pathogens, and also facilitate phagocytosis by cells of the innate immune system (e.g., monocytes and macrophages), and modulate immune responses [[81], [82], [83]]. The alveolar component also includes antimicrobial lysozyme and complement components [84], with the complement system believed to represent a key component of innate immunity against SARS-CoV-2 [85]. In addition, the lungs contain several types of resident immune cells (dendritic cells, macrophages, and mast cells) as well as itinerant immune cells (neutrophils, monocytes, and leukocytes) [86], with alveolar macrophages (AMs) being the predominant immune cells, accounting for 90% of airspace leukocytes [84] and involved in nonspecific phagocytosis of pathogens [87]. Several pathogen-associated molecular patterns (PAMPs) are exposed during virus replication and are recognized by pattern-recognition receptors (PRRs) expressed in respiratory epithelial cells, alveolar macrophages, and DCs, leading to the activation of signaling pathways that induce the production of type I and III interferon (IFNs) and inflammatory cytokines, which play a crucial role in the early combat of viral infections. [61,85,[88], [89], [90]]. For example, recent investigations have demonstrated that SARS-CoV-2 is susceptible to IFN-α or IFN-β treatment, suggesting that human type I IFN may be effective in suppressing SARS-CoV-2 infection [91].

2.2.2. Adaptive immune response

In addition to innate immunity, effective adaptive immune responses are essential for preventing respiratory viral infections [92]. On one hand, the induced antigen-specific cytotoxic T lymphocytes (CTLs) could specifically recognize and kill infected host cells. On the other hand, the induced antigen-specific antibodies by B cells could specifically neutralize viral particles. Fig. 2 depicts a schematic representation of the adaptive immune response in the mucosal immune system. To successfully induce the adaptive immunity mentioned above, antigens need to be first taken up by antigen-presenting cells (APCs), such as DCs [53]. The antigens are then processed and presented by the APCs to T cells in the T-cell region of the MALT [48]. Subsequently, these activated T cells proliferate and differentiate into CD8⁺ cytotoxic T cells that are capable of killing infected cells, and CD4⁺ T helper cells that help B cells become antibody-producing plasma cells [93]. The activated CD8⁺ T cells proliferate and obtain effector functions, including the production of cytokines and the cytotoxicity to eliminate respiratory viral infections [94,95]. The primary function of CD4⁺ T cells is to support the activation and differentiation of B cells for antibody production. The activated CD4⁺ T cells could be subdivided into Th1, Th2, Th17, and Treg subsets. Th1 cells further promote T cell immune responses and are the prerequisite for clearance of intracellular infections [78]. Th2 cells produce interleukins (IL-4, IL-5, IL-13) to induce humoral immunity. B cells are stimulated by these cytokines produced by Th2 cells (e.g., IL-4, IL-5, IL-6, IL-10, etc.) and differentiate into antigen-specific IgA⁺ B cells [96,97]. Due to tissue-specific homing mechanisms, antigen-specific IgA⁺ B cells migrate toward effector sites and differentiate into plasma cells that produce monomeric forms of IgA before forming dimers or polymeric IgA [53]. The monomeric IgA is then linked together by J-chains to form polymeric IgA (pIgA) [53,98], which forms the complex with polymeric Ig receptor (pIgR) expressed by epithelial cells for further translocation to the lumen surface. Some of the pIgR is digested on the luminal side [99], ultimately leading to the formation of secretory IgA.

Fig. 2.

Adaptive immune responses in the mucosal immune system. Antigens are processed and presented to T cells by APCs, which triggers their activation, differentiation, and expansion, ultimately mediating cellular responses (CTLs), humoral responses, and mucosal responses (sIgA). The figure was reproduced with permission from ref. [100] under the CC BY-NC-ND license. Copyright 2022 Elsevier.

The crucial characteristics of mucosal adaptive immune responses include the local synthesis and secretion of IgA antibodies and the production of tissue-resident memory T cells (T_RM) [101]. Secretory IgA acts prior to the onset of epithelial infection to promote the capture and removal of harmful pathogens or antigens in mucus by three mechanisms: antigen excretion, immune exclusion, and intracellular neutralization [[102], [103], [104]], ultimately preventing infections from invading mucosal areas [68,105,106]. In addition, dimeric sIgA possesses a higher affinity than monomeric IgG, resulting in a greater ability to neutralize SARS-CoV-2 [107,108]. It has been reported that mucosal IgA antibody responses were generated in the conjunctival and nasal fluids of adults and children within four days after infection with SARS-CoV-2 [109]. T_RM reside in the upper and lower airways and also play a crucial role in the prevention of respiratory viral infections. T_RM act rapidly and exert quick effector functions when pathogens are re-encountered [96,110]. They provide immediate tissue protection by recruitment of circulating memory T cells and production of inflammatory cytokines/chemokines [111,112]. Early research in mice has shown that CD4⁺ T_RM in the airways and CD8⁺ T_RM in the parenchyma could protect against SARS-CoV-1 [113,114]. Recent investigations have also indicated the presence of CD4⁺ and CD8⁺ T_RM in the pulmonary of patients with SARS-CoV-2 infection, which have been linked to reduced disease severity [115,116]. More importantly, these T_RM persisted in convalescent patients for at least ten months [117]. These results suggest that airway vaccination which produces sIgA and T_RM may be more effective in preventing SARS-CoV-2 infection than peripheral immunization.

3. Intranasal vaccines

The initial infection with SARS-CoV-2 occurs in the upper respiratory tract, making intranasal vaccines a focal point of research in the development of mucosal vaccines for COVID-19. In order to develop safe and efficacious nasal vaccines, it is important to carefully consider various aspects of the vaccine formulation, including the dosage form, antigen, mucosal adjuvant, delivery system, and excipients to overcome delivery barriers and trigger specific adaptive immune responses.

3.1. Intranasal vaccine formulations

There are several dosage forms of nasal vaccines currently in development, including gels, powders, drops, aerosol sprays, and solid inserts [[118], [119], [120]]. Among the licensed human intranasal vaccines are the influenza vaccines FluMist/Fluenz™ and Nasovac™, both of which are administered via intranasal spray. Liquid formulations are favored as they are easy to prepare, can be delivered to patients more conveniently, and are compatible with simpler devices [121]. Several studies have proven the efficacy and safety of intranasal administration of liquid vaccines. For example, FluMist® is the first approved human nasal spray vaccine that can be stored and shipped at 2–8°C, which does not require freezing, overcoming the challenges posed by the expensive cold chain. Other vaccines for intranasal delivery have been extensively investigated for autoimmune diseases such as arthritis [122] and infectious diseases including measles, meningitis, pneumonia, and tuberculosis, with the majority of these vaccines also in liquid form [[123], [124], [125], [126], [127], [128], [129]]. Similarly, several of the COVID-19 vaccine candidates currently undergoing clinical research for intranasal administration are liquid formulations.

The development of nasal vaccines has been hindered by various challenges due to the unique nature of the nasal environment. Firstly, the volume of nasal vaccine that can ultimately be used is limited (25–200 μL) [119], as any volume exceeding the capacity of the nasal cavity would be expelled from the nose [130]. Secondly, the residence time of nasal vaccines in the nasal cavity is limited due to mucociliary clearance [131,132]. For powder or liquid formulations, the half-life of clearance is usually within 15-20 minutes [133,134]. This short residence time may cause inadequate absorption of antigens by APCs in the NALT. In addition, the epithelial cells on the luminal surface of the respiratory tract are tightly connected, forming a mechanical-physical barrier [43,135], and most antigens also have a low affinity for epithelial cells [136], which would lead to inefficient uptake of antigens and may limit the efficacy of nasal vaccines [137]. Moreover, the antimicrobial proteins and the enzymatic environment in the respiratory tract may degrade vaccine components and affect the stability of the vaccine [135,138]. To overcome these obstacles and enhance the efficacy of nasal vaccines, various strategies have been developed, including the utilization of mucoadhesives, permeation enhancers, enzyme inhibitors, and vaccine adjuvants that boost the immunogenicity of antigens [130].

3.1.1. Mucoadhesives

Mucoadhesives have been demonstrated to extend the residence time of vaccines on the nasal mucosa and slow down mucociliary clearance [139]. Generally, mucoadhesives can be classified into three categories based on their mode of action. The first group consists of cationic polymers such as chitosan, chitosan-derived polymers, and cationic lipids, which interact with the negatively charged mucus glycoproteins of the nasal mucosal tissue through ionic interactions [140]. Notably, chitosan has been shown to interrupt the tight connections between epithelial cells [141], thereby enhancing the epithelial permeability and contributing to the effective uptake of antigens. It has been shown in several studies that chitosan is a prospective excipient for intranasal vaccinations, enhancing the antibody response to intranasal vaccines as a result of its mucoadhesive and adjuvant properties [[142], [143], [144], [145], [146], [147], [148]]. The second group of mucoadhesives is composed of hydrophilic polymers that prolong the nasal residence time by forming hydrogen bonds to mucus, including carbopol, sodium alginate, hydroxypropyl methylcellulose, and sodium carboxymethylcellulose [136]. The third group of mucoadhesives consists of thiolated polymers and thiomers [149], which possess strong mucoadhesive properties due to their capability to form disulphide bonds with the cysteine groups in mucin [150,151]. The application of mucoadhesive materials to intranasally administered COVID-19 vaccines has been shown to induce wide immune responses and the production of mucosal IgA and IgG [152]. For instance, Jearanaiwitayakul et al. exploited the mucoadhesive property of chitosan to develop an intranasal SARS-CoV-2 vaccine [153]. The RBD of SARS-CoV-2 spike glycoprotein was encapsulated as the immunogen in N, N, N-trimethyl chitosan nanoparticles (RBD-TMC NPs), and then immunized intranasally to mice. The results demonstrated that intranasal inoculation of TMC elicited robust mucosal immunity while also stimulating systemic humoral and cell-mediated immune responses in mice, in contrast to the nasal vaccine without TMC, which was unable to induce the production of IgA.

3.1.2. Permeation enhancers

The utilization of permeation enhancers is a widely recognized approach to facilitate the absorption and bioavailability of mucosal vaccines. Despite the mechanism of action of permeation enhancers has not been adequately clarified, it is widely accepted that their ability to disrupt tight junctions of the epithelium and enhance paracellular motility increases the permeability of the mucosa to the antigens [154]. A number of compounds have been identified as exhibiting permeation enhancing properties, including bile salts, cyclodextrins, fusidate derivates, surfactants, fatty acids, phosphatidylcholine, laureth-9 sulfate, and cell-penetrating peptides (CPP) [131,140,155,156]. In addition, chitosan [157] and its derivatives, as well as bacterial toxins [158], such as cholera toxin (CT) and its closely related heat-labile enterotoxin (LT), have also been extensively explored as permeation enhancers. Specifically, chitosan disrupts the tight junctions of epithelial cells by mediating the translocation of tight junction proteins (claudins, ocludin, ZO-1) from the membrane to the cytoskeleton [141,159]. Similarly, bacterial toxins can cause disruption of the tight junctions by binding to tight junction components, directly modifying tight junction proteins, or altering the peri-junctional actin filaments [[160], [161], [162]]. Although the permeation-enhancing effect of chitosan has been previously reported, chitosan is soluble and active as a permeation enhancer only in acidic environments in the form of protonation [163,164], which limits its application. To overcome this constraint, research has focused on investigating chitosan derivatives with higher solubility. N-trimethy chitosan chloride (TMC) is a partially quaternized chitosan derivative that exhibits excellent water solubility over a wide pH range [164] and has superior properties of mucoadhesion and permeability [165,166]. Furthermore, TMC is considered to be relatively non-toxic [164,[167], [168], [169]]. For instance, it was found that TMC caused no tissue damage to the nasal mucosa of rats [170]. Another study also confirmed that local toxicity induced by nasal vaccination with TMC as the adjuvant was minimal [171]. As a result, TMC has been widely used in recent years for research on nasal vaccines [153,[172], [173], [174]].

3.1.3. Adjuvants

Adjuvants are substances added to vaccine formulations to enhance the immunogenicity of the antigen and improve the efficacy of the vaccine. The development of adjuvants for clinical use is a particularly slow process, and there are currently no adjuvants licensed for nasal vaccinations in humans. One strategy for developing adjuvants for mucosal vaccines is to employ adjuvants that have been approved for parenteral human vaccine use, such as aluminum salts, MF59, CpG 1018, the Adjuvant Systems AS0 adjuvants (AS01_B, AS03, and AS04), and lipid nanoparticles (LNPs) [130,175]. Among these, the use of aluminum hydroxide as a nasal mucosal adjuvant seems promising. Alhydrogel®, an aluminum oxyhydroxide, has been shown to have adjuvant activity in nasal mucosal vaccines, enabling adsorbed antigens to trigger specific systemic and mucosal immune responses [176]. Du et al. immunized mice with recombinant SARS-CoV-2 RBD using Alhydrogel® as the adjuvant by three different routes of administration (intramuscular, intranasal, and microneedle vaccination) [177]. They demonstrated that intranasal immunization elicited a robust humoral response and generated the greatest mucosal immunity.

Another strategy is to develop novel mucosal adjuvants, including toxin-based adjuvants, agonists of PRRs, cytokine adjuvants, etc.

3.1.3.1. Toxin-based adjuvants

Toxin-based adjuvants, such as cholera toxin (CT) and Escherichia coli heat-labile toxin (LT), can bind to ganglioside GM-1 receptors found on the epithelium to regulate immune responses [178,179]. They can act not only as permeation enhancers but also as potent mucosal adjuvants to stimulate mucosal and serum antibody responses [178,179]. Despite their potential benefits, the application of toxin-based adjuvants has been hampered by safety concerns. For example, the increased risk of Bell’s palsy is thought to be related to LT [180], which was the reason for the withdrawal from the market of the first commercial nasal virosomal influenza vaccine containing LT as an adjuvant [181]. This phenomenon appears to be the consequence of the transport of LT adjuvant to the central nervous system (CNS) after intranasal administration. Although the route of absorption from the nose to the brain is not fully understood, two possible pathways are currently considered. One way involves pathways in the cerebrospinal fluid, vasculature, or lymphatic system, and the other is absorption and transport to the brain via the olfactory or trigeminal nerves [182]. Specifically, the olfactory epithelium is the only part of the CNS exposed to the external environment, and the intranasal administration could directly access primary olfactory neurons that communicate with the olfactory bulb in the brain via their axons, resulting in direct nasal-to-brain delivery [183,184]. In addition, it has been shown that CT and LT accumulate in the olfactory nerve after intranasal vaccination in mice, in which they can elicit potentially neurotoxic responses [180]. Thus, concerns about whether intranasal vaccines can enter the brain and cause neurological disorders have been raised, and these cases reinforce the potential dangers of toxin-based adjuvants. Consequently, safer adjuvants based on CT and LT detoxification derivatives, such as CTA1-DD [185,186], LTh(αK) [187], LTK63 [188], and LTR72 [189], have been developed. For instance, CTA1-DD is a detoxified derivative of CT. Bernasconi et al. discovered that the combination of CTA1-DD and lipid nanoparticles enhanced the immunogenicity and protection of intranasal influenza vaccines considerably [186]. In another study, Pan et al. evaluated the safety and immunogenicity of an intranasal inactivated influenza vaccine containing the adjuvant LTh(αK) and found that the vaccine with LTh(αK) had significantly enhanced mucosal immunity compared to the non-adjuvanted control vaccine [190].

3.1.3.2. Agonists of PRRs

A class of adjuvants works by binding to PRR on cells of the innate immune system, including Toll-like receptors (TLR) agonists, STING agonists, c-type lectin receptors (CLR) agonists, retinoic acid-inducible gene 1 (RIG-1)-like receptors (RLR) agonists, nucleotide-binding oligomerization domain (NOD)-like receptors (NLR) agonists, etc. [[191], [192], [193], [194]]. The binding of these agonists to PRRs triggers an intracellular signaling cascade in innate immune cells, leading to the maturation of DCs, secretion of cytokines, and co-stimulatory signaling to Th cells [149].

TLR agonists represent a primary subgroup of mucosal adjuvants that have received considerable attention in research, including Pam3CsK4 (TLR2 agonist) [195], Polyinosine-polycytidylic acid (Poly I:C) (TLR3 agonist) [196,197], MPL (TLR4 agonist) [[198], [199], [200]], flagellin (TLR5 agonist) [201,202], and CpG-oligodeoxynucleotide (CpG-ODN) (TLR9 agonist) [203,204]. For example, Pam3CSK4 is a TLR2 agonist and works by activating TLR2 to generate Th2 and Th17 responses [205,206]. Intranasal immunization with Pam3CSK4 as the adjuvant for the RSV virosomes vaccine has been demonstrated to induce serum IgG antibody responses and mucosal antibodies to protect mice from RSV infection [195]. Lipopolysaccharide (LPS), a ligand of TLR 2 and TLR 4, is the main component of the outer membrane of Gram-negative bacteria. It has been widely explored as an effective immunostimulatory agent, but its potential toxicity limits its use in humans. Monophospholipid A (MPL), a low-toxicity derivative of LPS [198], is considered an effective and safe vaccine adjuvant and is included in the injectable vaccine adjuvants AS01, AS02, and AS04 [207]. Intranasal immunization of mice using MPL as the adjuvant with three different antigens led to increased serum antibodies and mucosal sIgA, which confirmed the mucosal adjuvant activity of MPL [199]. The other potential mucosal adjuvant is Poly (I:C), a synthetic analogue of dsRNA that is recognized by endosomal TLR3 and activates innate immune responses to produce IgA [208] and has been extensively investigated for nasal mucosal immunization. Recent research on rhesus macaques demonstrates that the combination of Poly (I:C) and CpG is an effective and safe nasal adjuvant for the COVID-19 subunit vaccine [209]. Furthermore, TLR7/8 agonist-based adjuvants, such as imidazoquinoline compounds, are used for intranasal immunization. Recently, Abhyankar et al. developed an intranasal vaccine against SARS-CoV-2 that comprises the TLR4 agonist GLA and the TLR 7/8 agonist 3M-052 as adjuvants, which trigger both antigen-specific mucosal and systemic responses after intranasal vaccination [210]. Although there have been encouraging results in mucosal adjuvants using different TLR agonists, the potential toxicity of these adjuvants is also a concern. For example, Poly (I:C) is considered to have the disadvantages of low stability and toxic effects [211,212]. Its toxicity is related to the capacity of its activation of the MAVS pathway [213]. It has also been suggested that Poly (I:C) may have serious effects on fetuses [214,215].

As secondary signaling molecules in bacteria, cyclic dinucleotides (CDN) like the cyclic diadenylate monophosphate (c-di-AMP) and the cyclic diguanylate monophosphate (c-di-GMP) are representatives of novel mucosal adjuvants [216]. Their mechanism of action involves the activation of the pathway known as the stimulator of the interferon gene (STING), which results in the generation of IFN-I and further elicits adaptive immune responses [217]. It has been suggested that lung surfactant-biomimetic liposomes encapsulating STING agonists (2’,3’-cGAMP) could work as a mucosal adjuvant for a universal influenza vaccine, triggering fast and efficient humoral and cellular immune responses, and exhibiting sustained cross-protection against influenza [218]. A recent study by Jearanaiwitayakul et al. demonstrated that the use of the STING agonist (cGAMP) as the adjuvant enhanced the immunogenicity of an intranasal spike SARS-CoV-2 vaccine candidate [219]. Furthermore, An et al. created an intranasal subunit vaccine against SARS-CoV-2 by encapsulating STINGa, cGAMP, or 2'3'-cGAMP in liposomes as the adjuvant, which elicits systemic and mucosal immunity [220].

3.1.3.3. Cytokine adjuvants

The exploration of cytokines as adjuvants in nasal vaccines has gained attention due to their ability to activate and modulate adaptive immunity. A number of cytokines have been investigated, including IL-1, IL-4, IL-12, IL-15, IL-18, type I interferon (IFN-α/β), IFN-γ, and IFN-λ [189,[221], [222], [223]]. For example, the IL-1 family cytokines have been utilized as adjuvants for recombinant influenza virus hemagglutinin (rHA) vaccines, which induce IgA and IgG antibodies after intranasal immunization [224]. Similarly, IL-15 has been utilized as the adjuvant for an intranasal vaccine against SARS-CoV-2 [209]. However, it should be noted that large and repeated doses of cytokines may lead to serious adverse effects [221].

3.1.3.4. Others

The saponins (Quil A, QS21) [225], the natural killer T (NKT) cell agonist(The α-galactosylceramide (α-GalCer))[226] and the protease-activated receptor 2 (PAR-2) agonist (PAR-2AP) [227] are promising intranasal mucosal adjuvants. In addition, polymers such as chitosan [228] and polyethyleneimine (PEI) [229,230] exhibit potential as mucosal adjuvants due to their delivery capacity and immunostimulatory effects. For instance, Lei et al. investigated three cationic nanocarriers, chitosan, N-[1-(2,3-Dioleoyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTAP) and PEI, and showed them to be effective adjuvants for the SARS-CoV-2 recombinant RBD vaccine by intranasal or intramuscular immunization [231]. Similarly, the safety of these adjuvants must be a concern of utmost importance. For example, the natural product saponin Quil A exhibits toxicity for human use, causing adverse effects such as severe local reactions, granulomas, and hemolysis [232]. Despite the development of a less toxic derivative, QS-21, similar safety concerns persist. Therefore, the safety of mucosal adjuvants would have to be thoroughly evaluated prior to widespread use.

3.1.4. Delivery systems for intranasal vaccines

The administration of intranasal vaccines requires effective delivery systems to protect antigens from degradation and promote cellular uptake, thereby enhancing their immunogenicity. The most commonly used polymers for the preparation of nasal vaccine delivery systems are polyester derivatives such as poly (lactic acid) (PLA), poly (lactic-co-glycolic acid) (PLGA), and their copolymers, as well as polysaccharides (such as alginate, chitosan, and dextran). For example, Kumar et al. designed a gold-nanostar-chitosan (AuNS-chitosan) as the carrier of antigen for intranasal administration of the SARS-CoV-2 DNA vaccine, which induced mucosal IgA, lung T_RM cells, and neutralized IgG responses in mice [233]. The specific discussion on nanocarriers is presented in the next section.

3.1.5. Devices for nasal administration

Liquids are the most widely used dosage form for administering nasal vaccines and medications. The traditional method of intranasal administration, such as through the use of pipettes, squeeze bottles, or droppers [234,235], has the disadvantage of not being standardized and precise. Therefore, new intranasal delivery devices, including spray devices and nebulizers, have been developed. The nasal cavity has unique physiological characteristics that affect the deposition of particulates. Large particulates are usually deposited in the anterior nasal portion, while particulates smaller than 10 μm in size have the potential to bypass the nasal cavity and enter the lungs [236]. As a result, it is essential for intranasal devices that deliver aerosols to achieve an optimal mean particle size (MPS) [234] in order to maximize the exposure of antigens in the nasal mucosa. In addition, the site of antigen deposition in the nasal cavity affects the absorption of intranasal vaccines. It is well known that the nose is divided into two nasal cavities through the midline septum, with each cavity consisting of three regions: the vestibule, the olfactory region, and the respiratory region [237]. The olfactory zone has the potential to transport drugs to the brain and is the targeted absorption region for drugs used in the treatment of central nervous diseases [134,238]. The respiratory zone, which occupies the main part of the nose and contains the superior, middle, and inferior turbinate bones, is the primary absorption area for systemic drug delivery [239]. For intranasal vaccine administration, the NALT region is the target deposition site for higher immunogenic responses, which includes the inferior, middle, and superior turbinate, and the nasopharyngeal region [121,240]. Metered-dose spray pumps are the most commonly used intranasal medication delivery systems on the market [241]. These devices typically deliver between 25 and 200 μL per spray, with an average dose of 100 μL, and can be adjusted for various sizes of particles and plume geometries [242]. For vaccines or expensive drugs, single-dose spray devices are often preferred. FluMist® is an approved human nasal vaccine administered by the AccuSpray Device (Fig. 3a), which consists of a disposable pre-filled syringe that produces large particles with diameters of 50 to 200 μm depending on the speed of the plunger [130,243]. These large particles can reduce vaccine deposition into the lower respiratory tract to minimize potential adverse effects. Spray devices offer several advantages, including portability, ease of use, self-administration, and safety. Nebulizers have also been utilized for intranasal administration. There are three primary categories of nebulizers based on the aerosol production technology: vibrating mesh nebulizers, ultrasonic nebulizers, and jet nebulizers (Fig. 3b) [244]. The MAD Nasal™ Device (Fig. 3c) is a painless intranasal drug delivery device developed by Teleflex, typically used to generate droplets which are then delivered to the nasal cavity in an atomized form [[245], [246], [247]]. The device has been used in numerous studies. For example, the MAD Nasal™ Device was utilized to administer the intranasal COVID-19 vaccine (ChAdOx1 nCoV-19) in a preclinical trial [248].

Fig. 3.

Spray and nebulizing devices for intranasal vaccine delivery. (a) The BD Accuspray™ nasal spray system. Adapted from ref. [249]. Contains information from the Government of Manitoba, licensed under the OpenMB Information and Data Use License (Manitoba.ca/OpenMB). (b) The three types of nebulizers and their mechanisms. Adapted with permission from ref. [250]. Copyright 2021 Taylor & Francis. (c) The Mucosal Atomizer Device (MAD) is used to deliver drugs through the nasal cavity with a fine spray. Adapted with permission from ref. [251] under the terms of the CC-BY 4.0 license. Copyright 2015 MDPI.

3.2. Intranasal COVID-19 vaccines under clinical investigation

The COVID-19 vaccines currently authorized for use are all administered intramuscularly. Given the advantages of vaccines administered via the respiratory tract in targeting respiratory pathogens, various respiratory-delivered vaccines against COVID-19 are also in development, with seven nasal COVID-19 vaccine candidates under clinical investigation, including four viral vector-based vaccines, two live attenuated vaccines, and one protein subunit vaccine (Table 2 ).

Table 2.

Intranasal SARS-CoV-2 vaccine candidates in clinical trials

Vaccine candidates	Platform	Developers	Dose	Schedule	Phase	Trial registries
ChAdOx1 nCOV-19 (AZD1222)	VVnr	University of Oxford	1-2	Day 0+28	I	NCT04816019
CVXGA1	VVnr	CyanVac LLC	1	Day 0	I	NCT04954287
BBV154	VVnr	Bharat Biotech International Limited	1	Day 0	III	CTRI/2022/02/040065
DelNS1-2019-nCoV-RBD-OPT1	VVr	Beijing Wantai Biological Pharmacy	2	Day 0+28	III	ChiCTR2100051391
COVI-VAC	LAV	Codagenix/ Serum Institute of India	1-2	Day 0+28	III	ISRCTN15779782
MV-014-212	LAV	Meissa Vaccines, Inc.	1	Day 0	I	NCT04798001
CIGB-669 (RBD+AgnHB)	PS	Center for Genetic Engineering and Biotechnology (CIGB)	3	Day 0+14+28 or Day 0+28+56	I/II	RPCEC00000345

VVnr: Viral Vector (non-replicating), VVr: Viral Vector (replicating), LAV: Live Attenuated Virus, PS: Protein subunit. Data from https://clinicaltrials.gov/, http://www.ctri.nic.in/, https://www.isrctn.com/ and https://www.chictr.org.cn/index.aspx.

Viral vector-based vaccines are considered a promising option among COVID-19 vaccines, and they can be further categorized into replicating and non-replicating (replication-deficient) viral vector vaccines. These viruses are harmless and serve as carriers of genetic information, which host cells utilize to produce antigens that initiate the body's immune responses [252]. A variety of viral vectors have been employed in COVID-19 vaccine development, including Modified Vaccinia Ankara (MVA) virus, Adenovirus, Influenza virus, Parainfluenza virus, and Rabies virus [253]. Adenoviruses are regarded as superior vectors for the delivery of target antigens to mammalian hosts, with the advantages of being simple to prepare and produce on mass scale, readily purified to high titers, genetically stable, and comparatively inexpensive [254,255]. In addition, adenovirus vector-based vaccines can be provided by multiple routes, including intramuscular, oral, intranasal, and intradermal [254]. Among the eleven COVID-19 vaccines approved by the WHO, four are based on viral vectors, and they are all adenovirus vaccines, including AZD1222, Covishield, Ad26.COV2.S, and Convidecia (Ad5-nCoV) [256]. Additionally, two intranasal adenovirus vector-based vaccines against COVID-19 are undergoing clinical trials.

ChAdOx1 nCOV-19 (AZD1222) is a replication-deficient simian adenovirus vector-based vaccine [257]. It has been reported that the effectiveness of the intramuscular Oxford-AstraZeneca vaccine against symptomatic COVID-19 was 81.3% [258], and the vaccine has been authorized by several medicine agencies worldwide, such as the WHO, FDA, EMA, etc. Previous research on rhesus macaques has demonstrated that intramuscular immunization with ChAdOx1 nCoV-19 does not diminish SARS-CoV-2 shedding from the upper respiratory tract [259]. Oxford/AstraZeneca continues to explore the potential of ChAdOx1 nCOV-19 for use as an intranasal vaccine. Intranasal delivery of this vaccine has been completed in hamsters and rhesus macaques with positive results, confirming that intranasal administration of the ChAdOx1 nCov-19 vaccine led to a significant reduction in viral load in the respiratory tissues of animals, providing almost complete protection [248,260,261].

Another intranasal COVID-19 vaccine candidate is ChAd-SARS-CoV-2-S. This vaccine uses the chimpanzee adenovirus vector that encodes the pre-fusion stabilized spike protein (S protein) [262]. Bricker et al. demonstrated that the ChAd-SARS-CoV-2-S nasal vaccine produced robust neutralizing antibody responses to SARS-CoV-2, effectively protecting Syrian hamsters from respiratory infections and providing better protection than intramuscular injections [263]. Additionally, research conducted on rhesus macaques has demonstrated that intranasal vaccination prevents upper and lower respiratory tract infections [15,264]. The vaccine, also known as BBV154, underwent a phase I clinical study to assess its reactogenicity, immunogenicity, and safety.

In addition to adenovirus vector-based vaccine candidates, vaccine candidates based on the canine parainfluenza virus (PIV5) are also making progress. CVXGA1 is an intranasal spray vaccine developed by CyanVac and its subsidiary Blue Lake Biotechnology, which is based on an attenuated strain of PIV5 and encodes the spike (S) protein of SARS-CoV-2. The efficacy of the intranasal vaccine against SARS-CoV-2 was demonstrated by An et al., who showed that a single dose of intranasal vaccination with CVXGA1 prevented lethal infection in K18-hACE2 mice and also protected against viral infection and contact transmission in ferrets [265].

Influenza viruses have also been utilized as significant viral vectors for vaccines. The COVID-19 vaccine, named DelNS1-2019-nCoV-RBD-OPT1, was developed by Beijing Wantai Biological Pharmacy and is administered as a nasal spray. Against stimulate human immune responses to SARS-CoV-2, the vaccine employs a replicating viral vector platform based on the influenza virus vector expressing the RBD of the SARS-CoV-2 spike protein [266]. Phase I and II clinical trials of this vaccine have been registered in China to assess its safety and immunogenicity. Phase III clinical trials to assess the vaccine's protective efficacy against symptomatic COVID-19 are also underway.

In addition to viral vectors, live attenuated vaccines represent a crucial category of intranasal COVID-19 vaccine platforms. Live attenuated vaccines contain live viruses that have been weakened [267]. Generally, these strains are attenuated mainly through adaptation to cold culture conditions, rational gene modifications, or through non-human animals [255,268], and they proliferate slowly in the human body, inducing strong immune responses without causing severe disease in healthy individuals. Live attenuated vaccines currently in use include measles, mumps, rubella (MMR), influenza, shingles, varicella, and polio vaccines, etc. Live attenuated COVID-19 vaccines for intranasal administration are also in development, and two of them are currently in clinical trials, which are COVI-VAC and MV-014-212.

COVI-VAC is a single dose intranasal COVID-19 vaccine. This vaccine is a live-attenuated SARS-CoV-2 based vaccine with the identical structural design and the same amino acid sequence as wild-type SARS-CoV-2 [262], but the coding region of the spike protein has been recoded and the furin cleavage site removed [269], which slows down its replication and increases its safety. Wang et al. evaluated the efficacy and safety of the vaccine in golden Syrian hamster models [269]. The findings indicated that hamsters inoculated intranasally with COVI-VAC developed significant levels of neutralizing antibodies equivalent to those in SARS-CoV-2-infected animals, indicating that COVI-VAC is effective in preventing infection with SARS-CoV-2.

Another live attenuated COVID-19 vaccine administered intranasally is MV-014-212. This is an adjuvant-free single-dose vaccine developed by Meissa Vaccines, Inc., based on the live attenuated recombinant human respiratory syncytial virus (RSV) expressing a chimeric SARS-CoV-2 spike as the only viral envelope protein [266,270]. Preclinical research conducted by Tioni et al. has demonstrated that MV-014-212 is both attenuated and immunogenic in non-human primates, leading to the development of both mucosal and systemic immunity after a single mucosal immunization. The results of this research indicate that the vaccine provides highly protective effects against SARS-CoV-2 in both the upper and lower respiratory tract [270].

Subunit vaccines are also promising intranasal COVID-19 vaccine platforms. Subunit vaccines contain purified fragments of pathogens, such as proteins, polysaccharides, peptides, or viral fractions that may form virus-like particles (VLPs) [271] and therefore have the capability to stimulate immune cells. As these fragments do not have the ability to cause disease, subunit vaccines are regarded as safe. However, these antigens are often insufficiently immunogenic to elicit protective immunity, thus adjuvants are required to potentiate immune responses [272]. Depending on the principal target antigens, SARS-CoV-2 protein subunit vaccines could be roughly divided into three categories: S protein-based vaccines, RBD-based vaccines, and VLP vaccines [255]. As of December 2022, protein subunit vaccines are the most widely researched vaccine platform against COVID-19, and there are presently 55 protein subunit vaccines against COVID-19 in clinical development, such as Novavax, Corbevax, Soberana 02, and Zifivax, etc. [3]. To date, the only intranasal protein subunit COVID-19 vaccine candidate in clinical trials is CIGB-669 (RBD+AgnHB). This vaccine was developed by the Center for Genetic Engineering and Biotechnology (CIGB) and consisted of the RBD protein and the HBV nucleocapsid (N) antigen (AgnHB, an immunopotentiator) [229]. Phase I/II clinical trials for this vaccine have been conducted in Cuba.

4. Pulmonary vaccines

Pulmonary vaccination is an attractive strategy for the protection of populations, particularly against diseases of the respiratory tract. Pulmonary vaccination has been widely used to immunize poultry since 1952, when an aerosol Newcastle disease vaccine was used in chickens [[273], [274], [275]]. However, the majority of research on pulmonary vaccines for human use remains in the preclinical stage, with only a limited number of vaccines having been tested in clinical trials. The development of pulmonary vaccines presents several challenges, including the need to overcome physiological barriers in the respiratory tract, ensuring vaccine stability, and selecting appropriate delivery systems and devices. Therefore, the design of safe and effective formulations that can overcome these barriers to pulmonary vaccine delivery is imperative.

4.1. Pulmonary vaccine formulations

4.1.1. Formulation considerations

The physicochemical parameters of the delivered antigen play a crucial role in determining its efficacy. These properties include the shape, size, surface charge, and hydrophobicity of the particles.

Firstly, the size of the aerosol droplet or particle has a significant impact on the area of the lung in which the delivered antigen will be deposited. Typically, particle sizes between 0.5 and 5 μm are deemed optimal for lung deposition [276]. The lungs consist of a complex network of airway tissues known as the bronchial tree, and particulates are deposited in the respiratory tract through three mechanisms: inertial impaction, sedimentation, and Brownian diffusion [277]. In general, large particles (>10 μm diameter) tend to be deposited in the oropharynx, whilst particles with sizes between 5 and 10 μm are deposited in the upper respiratory tract, limiting the quantity of medication delivered to the lungs. Particles with diameters of 0.5-5 μm are deposited in the alveoli and tiny airways. Additionally, particulates smaller than 0.5 μm are generally not deposited but are expelled during exhalation [276,278].

Secondly, these physicochemical properties affect the interaction between vaccine antigens and APCs. The APCs in the human lung are composed of various cell types, including alveolar macrophages, DCs, and B cells. Notably, alveolar macrophages have higher phagocytic activity but less potency in presenting antigens, while DCs have higher APC capacity [41,279,280]. Pathogen-sized particulate antigens can imitate the typical uptake by APCs and induce effective immune responses [41]. It has been demonstrated that 50 nm particles are more effectively taken up by DCs in the lung mucosa and induce co-stimulatory signals than 500 nm particles, which were mainly taken up by alveolar macrophages, as DCs and macrophages take up particles of different sizes. Also, particle shape plays a role in cellular uptake. Kumar et al. demonstrated that small spherical particles with average diameters of around 190 nm elicited Th1-biased immune responses, whereas larger rod-shaped particles with average diameters of approximately 1.53 μm elicited Th2-biased immune responses [281]. In another research, Sharma et al. investigated the immune response to antigens with different shapes of PRINT particles. They discovered that the antibody titer of particles resembling earthworms was higher than that of particles with cylindrical, hexagonal, or cuboidal particles [282].

In addition to the size and shape of the particles, the uptake of vaccine antigens can also be influenced by the surface charge. Theoretically, the mucus layer and the cell membrane are negatively charged. and thus positively charged particles are thought to interact with them more effectively than negatively charged particles, leading to an increase in residence time and cellular uptake [283]. It has been found that particles with a negative surface charge decrease the uptake by APCs [284]. Fromen et al. conducted a study to assess the effect of nanoparticle charge on systemic and mucosal antibody responses after lung administration, keeping the charge as the sole variable and holding all other physical and chemical parameters constant [285]. Their results showed that positively charged nanoparticles could elicit robust systemic and mucosal antibody responses after lung immunization, while negatively charged nanoparticles were ineffective. They further explored the mechanism of action of nanoparticles charge on pulmonary APCs association and demonstrated that cationic nanoparticles interacted preferentially with two critical lung DC populations [286]. Cationic nanoparticles not only promoted DC association but also regulated the local lung environment to facilitate recruitment and maturation of lung DCs while preventing substantial clearance of AMs.

Moreover, the uptake and transportation of particles in the lung can be affected by surface hydrophobicity. Hydrophobic interactions act by influencing the mucus diffusion of the nanoparticles [287]. The mucus consists mainly of water and is predominantly hydrophilic. As a result, hydrophilic particles do not form powerful adhesive bonds with mucus, their interactions are weakened, and molecules are able to freely penetrate the mucus layer, hence performing better in terms of mucus penetration and exhibiting better retention behavior compared to hydrophobic nanoparticles [287,288]. Schneider Craig et al. compared the retention behavior of PLGA nanoparticles with or without hydrophilic PEG coating and found that PLGA-PEG nanoparticles were retained in the lung cavity for a longer duration and had a lower clearance in the lung after 2 hours [289].

The administration of inhaled vaccines is subject to pulmonary clearance mechanisms, including mucociliary clearance in the conducting airways, clearance by alveolar macrophages, and enzymatic degradation [276,290]. Similar to intranasal formulations, the use of permeation and absorption enhancers, mucoadhesives, and enzyme inhibitors are potential ways to improve pulmonary absorption. A variety of excipients have been explored as aerosol penetration and absorption enhancers, mainly including bile salts (sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate) [291,292], phospholipids (dipalmitoylphosphatidylcholine, phosphatidylcholine, phosphatidylglycerol) [293], fatty acids (arachidonic, capric and lauric acids), non-ionic surfactants (poloxamer 188), and bio-surfactants (rhamnolipids) [294]. For instance, research by Thomas et al. showed that alkylglycosides, the non-ionic surfactants, could enhance the nasal and pulmonary absorption of different drugs [295,296]. Furthermore, in a rodent model, they showed that the use of tetradecyl-β-maltoside (TDM) formulated with the hepatitis B vaccine enhanced immune responses after pulmonary administration [297]. The mechanism of action of these absorption enhancers involves disruption of epithelial tight junctions, extraction of membrane proteins or lipids, and membrane fluidization, thereby perturbing the cell membrane [292,294]. However, this may result in irreversible damage to the alveolar epithelial cell layer [298,299], raising safety concerns regarding the use of absorption enhancers in pulmonary vaccines. For instance, despite the potential of bile salts as absorption enhancers, their toxicity limits the application. Bile salts have been reported to disrupt surfactant function in vitro and induce irritation of the pulmonary in vivo [300]. Additionally, alkylglycosides exhibit remarkable toxicity to the epithelial cells of the airway [301]. Thus, the safety of absorption enhancers used in pulmonary vaccines must be thoroughly evaluated.

In addition to overcoming the physiological barrier of the lung, stability is a crucial consideration for vaccine formulations. Liquid formulations are the most common used dosage form of vaccines, but they are relatively unstable, require strict storage and transport conditions, and may also be subject to shear stresses during nebulization. To enhance stability, one strategy is to adjust the osmotic pressure and pH of the formulation, as well as the ionic strength of the buffering salts [241,302]. In general, vaccines maintain optimal activity within a pH range of 5 to 8 and exhibit increased stability at low osmolarity and comparatively low ionic strength of buffering salts (I < 0.15) [303]. Sodium hydroxide, hydrochloric acid, phosphates, and citric acid are commonly used to adjust pH, while sodium chloride is utilized to regulate osmolality in vaccine formulations [304]. Another solution to enhance stability is the addition of stabilizing excipients [24]. Compatible osmolytes and surfactants are widely used as stabilizing excipients [24]. The theory of preferential hydration is the principle by which compatible osmolytes act as stabilizers [24,305,306], which implies that when osmolytes (stabilizing agents) are added to the solution, water molecules preferentially interact with the protein and osmolytes are preferentially excluded from the protein surface [[307], [308], [309]]. Common compatible osmolytes include l-serine, glycine, and trimethylamine N-oxide [24]. Surfactants are also commonly employed as stabilizers in protein formulations, which act by reducing surface tension, decreasing the free energy of the system, and reducing protein-interface interactions as well as protein-protein interactions [310]. Specifically, surfactants outperform protein molecules in their ability to adsorb to hydrophobic surfaces (e.g., air/water interfaces), thereby preventing proteins adsorption to these hydrophobic interfaces from generating aggregates [311,312]. For example, phosphatidylcholine, including egg phosphatidylcholine (EPC), lysophosphatidylcholine (LPC), distearoylphosphocholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), tyloxapol, Tween 20, Tween 80, Pluronic F127, octaethylglycol, and caprylic/capric glyceride, have been employed in solution or particulate suspension formulations [313].

The dry powder formulation, a more stable dosage form than the traditional liquid formulation, has become an increasingly popular area of research in pulmonary vaccine development. Optimal particle sizes for lung deposition are in the range of 0.5-5 μm [278], and a number of advanced manufacturing technologies have been used to produce these respirable aerosol nanoparticles, including spray drying, spray freeze drying, particle replication in nonwetting templates (PRINT), supercritical fluid technology, and thermal condensation using capillary aerosol generator [314]. Freeze drying is a common drying method used in pharmaceutical industries and consists of three main steps, which are freezing (solidification), primary drying (ice sublimation), and secondary drying (desorption of unfrozen water) [315]. As the formulation is subject to ice crystal formation and stress during freezing and drying steps [41,316], stabilizers or cryoprotectants are often added to ensure the stability of the formulation. Polyols and sugars (e.g., glucose, inulin, sucrose, lactose, trehalose, mannitol, sorbitol, myo-inositol) are commonly used as cryoprotectants [24,317]. Once in the dry condition, the sugar matrix also provides remarkable stability for storage of the formulation through a mechanism known as vitrification [318]. However, freeze drying produces cake-like structured material with uncontrollable particle properties [41,79]. Therefore, freeze-drying may be less suitable for the manufacture of inhaled dry powder vaccines, and a range of alternative drying methods have been developed. Spray drying may be an effective alternative method and is the technology for converting liquid feed into potentially respirable dry particles, involving four main stages: liquid feed atomization, droplet drying, powder collection, and subsequent processing [41,315]. Similarly, glass-forming excipients such as sugars, polyols, and organic acids are incorporated to enhance the formulation's stability during processing and storage [79]. Leucine is also typically added to spray drying feed solutions or suspensions as a dispersion enhancer to improve the dispersibility of dry powders for pulmonary delivery and to prevent moisture-related degradation and loss of aerosol properties [319,320]. Sou et al. proved that spray drying influenza antigens with trehalose and leucine as excipients could produce a highly nebulized powder vaccine formulation that induced robust mucosal and systemic protection following pulmonary immunization [321]. Spray freeze drying is the combination of spray drying and freeze drying, which has the advantage of producing size-adjustable particles and insulates the formulation from thermal stresses [79,316,322]. Audouy et al. prepared an influenza whole inactivated virus vaccine in dry powder form using the spray-freeze drying technique with inulin as a cryoprotectant, which produced a good systemic response after pulmonary immunization in mice and proved to be safe and effective [323].

4.1.2. Delivery systems for pulmonary vaccines

Nanoparticles are widely employed as delivery systems in vaccines. Delivery systems are also considered to be adjuvants that have the advantages of being more readily recognized by antigen presenting cells than soluble antigens [24], having immunostimulatory properties, and significantly enhancing immune responses [324]. Additionally, nanoparticle delivery systems have been used to overcome respiratory barriers such as mucociliary clearance, enzymatic degradation, and phagocytosis by alveolar macrophages, t thus resulting in an increase in drug uptake within the respiratory system [325].

The delivery systems commonly used for pulmonary vaccines include polymeric nanoparticles and lipid-based nanoparticles. Polymers can be further classified into two categories depending on the source material: natural or synthetic. Chitosan, a natural polysaccharide polymer, is positively charged under physiological conditions and therefore exhibits mucoadhesive properties [41]. In addition, chitosan has permeation properties, high biocompatibility and biodegradability [44], which make chitosan and its derivatives (e.g., trimethyl chitosan, hydroxyethyl chitosan, and sulphated chitosan) attractive polymers for mucosal vaccine delivery [326]. A recent study by Zhuo et al. designed an inhalable nanovaccine containing SARS-CoV-2 spike protein and chitosan, which produced robust spike-specific antibody immune responses and enhanced local mucosal immunity in bronchoalveolar lavage and lungs, potentially protecting the host against SARS-CoV-2 infection without systemic toxicity [327]. Other natural polymers, including gamma polyglutamic acid, hyaluronic acid, pullulan, alginate, and gelatin, have been employed in nanoparticle-based mucosal vaccine delivery systems. Synthetic polymer-based nanoparticles have also been widely explored, including poly (glycolic acid) (PGA), poly (lactic acid) (PLA), PLGA, PEI, polyphenylene sulfide (PPS), and poly-ε-caprolactone (PCL). PLGA is one of the most successfully developed polymers. The release of antigens and adjuvants by PLGA nanoparticles is controlled and sustained over weeks or months, which stimulates effector T cell memory responses [328]. Moreover, PLGA is biodegradable and biocompatible, and the FDA and EMA have authorized its usage in parenteral medication delivery systems [329]. Muttil et al. designed a novel NP-aggregate formulation utilizing recombinant hepatitis B surface antigen (rHBsAg) and PLGA and demonstrated that the dry powder formulation triggered strong mucosal immune responses in guinea pigs following pulmonary administration [330].

In addition to polymers, liposomes are widely researched as pulmonary delivery systems and are spherical vesicles consisting of one or more phospholipid membranes wrapped around an internal aqueous core [329,331]. The composition and physicochemical properties of liposomes are tailorable [44] and could be utilized to deliver a broad variety of substances, including DNA, RNA, proteins, and peptides [41]. Biocompatible neutral and anionic phospholipids, such as cholesterol and phosphatidyl cholines (e.g., DSPC), are the most prevalently employed components of liposomes [331]. Liposomal vaccines could be formulated as dry powders or liquids. Zhang et al. have designed a nebulisable lipid nanoparticle that holds promise for the pulmonary delivery of mRNA [332]. Further lipid-based nanocarriers for pulmonary vaccine delivery are also being investigated, including solid-lipid nanoparticles (SLN) [276], immunostimulatory complexes (ISCOMs) [41,333], gas-filled microbubbles (GFM) [44], archaeosome [78], virus-like particles, and virosomes [44]. For instance, Liu et al. prepared a phosphatidylserine-coated liposomal mucosal nanovaccine which loaded with the STING agonist (cGAMP) (NP-cGAMP) that could target APCs in the lungs and enhance anticancer immunity against lung metastases after inhalation administration in a mouse model [334]. Furthermore, Zheng et al. have designed a bionic-virus nanovaccine that uses the RBD of SARS-CoV-2 as the antigen, the Poly (I:C), which mimics the genetic substances of the virus, as the immune adjuvant, and the pulmonary surfactant (bio-PS) liposome, which mimics the structure of the viral capsid, as the delivery vector. This mimic virus-like structure of the inhalable nanovaccine provides stronger mucosal protection than intramuscular and subcutaneous vaccination, effectively neutralizing the virus and preventing its entry into the body via the respiratory system [335].

4.1.3. Devices for pulmonary administration

For the pulmonary delivery of vaccines, the dispersion of the device has to produce an inhalable fine aerosol with aerodynamic properties appropriate for pulmonary administration, which typically requires the size of particles aerodynamically in the range of 0.5-5 μm. The optimal site of deposition for pulmonary vaccines is not completely known, and studies have shown conflicting results, which may be associated with the diseases targeted by the vaccine. For example, Minne et al. delivered a monovalent influenza vaccine to different respiratory regions of mice, including the nasal cavity, upper airway, central airway, and deep lung, to explore the effect of the delivery site on the immune response [336]. The results indicated that the deep lung was the optimal target for inhaled influenza vaccination. However, in another study in cotton rats, it was shown that the influenza vaccine produced comparable protection when deposited in the trachea and lungs [337]. The site of deposition during pulmonary delivery of the influenza vaccine had no significant effect on the protective efficacy of the vaccine. In addition, Tomar et al. evaluated the effect of deposition sites in the respiratory tract on two vaccines with different efficacy and found that tracheal/central airway and deep lung targeting induced comparable immune responses when delivering influenza vaccine, whereas immune responses were considerable following deep lung deposition and almost absent following tracheal/central airway deposition when delivering hepatitis B vaccine [338]. They concluded that the site of deposition in the lung had a minor correlation with the influenza vaccine, while deep lung deposition was significant for the hepatitis B vaccine. Thus, the optimal site of deposition of the vaccine in the pulmonary may be related to the disease targeted by the vaccine. The site of lung deposition is less influential for vaccines against infectious diseases transmitted through the respiratory tract, while it is critical for vaccines against systemic viral infections (e.g., hepatitis B). If this hypothesis proves correct, it would be extremely beneficial for devices used for pulmonary delivery of the COVID-19 vaccine that may not necessarily require strict implementation of deep lung deposition.

There are three main categories of inhalers that are widely used today: nebulizers, pressurized metered-dose inhalers (pMDIs), and dry powder inhalers (DPIs) [313].

Nebulizers are intended to break down aqueous solutions or suspensions into aerosols for administration. For example, the Classic Mexican Device (Fig. 4a), a well-known nebulized vaccine delivery device invented in the 1980s [130], has been used to administer aerosolized measles vaccination to children in mass campaigns in Mexico and in several clinical trials [[339], [340], [341]]. The Aerogen Ultra device (Fig. 4b) is a nebulizer that produces a large number of small homogeneous particles with vibrating net technology and was used in the phase I clinical study to nebulize the COVID-19 vaccine (Ad5-nCoV) for aerosol administration [342]. However, it is essential to consider the detrimental effects of nebulizers on large molecules (e.g., antigenic proteins), including the shear forces generated during nebulization [[343], [344], [345]], protein aggregation due to the increased air-liquid interface [[346], [347], [348]], and the heat generated during ultrasonic nebulization [344,346,348]. For example, it has been demonstrated that ultrasonic nebulizers, which generate aerosols through high frequency vibrations, could induce protein aggregations and inactivation [24,349,350]. Additionally, another investigation has indicated that considerable degradation of model proteins would occur during jet and ultrasonic nebulization [351]. Furthermore, ultrasonic nebulizers and jet nebulizers have a large dead volume and may cause up to 30% waste of the vaccine [24,346,348]. The pMDI can also be used to deliver liquid solution or suspension formulations. However, its propellants are typically deemed inappropriate for use as dispersants in biological products, such as vaccine formulations. It has been reported that pMDI could produce small particle aerosols for bacterial vaccines, but with a substantial loss of antigenicity [352]. Given these factors, dry powder inhalers (DPIs) are considered to be the most promising delivery device for pulmonary vaccines.

Fig. 4.

Nebulizers for the delivery of pulmonary vaccines. (a) Diagram of the classical Mexican device for aerosolizing liquid measles virus vaccines. Reproduced with permission from ref. [357]. Copyright 2014 Taylor & Francis. (b) The Aerogen Ultra device. Adapted from ref. [358].

Dry powder inhalers are portable and compact devices that are available as single-dose or multi-dose devices and eliminate the cold chain requirement. However, the lung deposition depends on the patient's inhalation flow and force, making it unsuitable for all age groups (e.g., infants) [79]. Some commercially available DPIs include the Aerolizer® (single dose device) (Fig. 5a), Diskus® (multiunit dose device) (Fig. 5b), Turbuhaler® (reservoir type system) (Fig. 5c) etc. [313,353]. The research on dry powder vaccines for pulmonary delivery is still limited, with only one clinical trial of pulmonary delivery of vaccines using the DPI device reported to date [354]. The study involved dry powder measles vaccination in adult males using the Puffhaler (Aktiv-Dry) (Fig. 6a) and Solovent (BD Technologies) (Fig. 6b) devices. These two devices have proven successful in rhesus macaques studies, where a single dose of dry powder measles vaccine elicited sustained and completely protective immunity [24,355]. In addition, the SOLO inhaler (Fig. 6c), TwinCaps (Fig. 6d), and Occoris (Fig. 6e), are considered to be disposable DPIs with significant potential to deliver inhalable vaccines [356].

Fig. 5.

Types of dry powder inhalers (airflow shown by arrowed streamlines, medication powder mixture indicated by dots). The figure was reproduced with permission from ref. [356]. Copyright 2022 Elsevier.

Fig. 6.

Examples of DPI for pulmonary vaccination. (a and b) Devices used to administer dry powder measles vaccines. Puffhaler (a) and BD Solovent (b) inhalers. The figure was reproduced with permission from ref. [355]. Copyright 2011 National Academy of Sciences. (c, d, and e) Three disposable DPIs. (c) SOLO; (d) TwinCaps; (e) Occoris. Adapted from ref. [356,[359], [360], [361]].

4.2. Pulmonary COVID-19 vaccines under clinical investigation

Currently, there are three inhaled COVID-19 vaccine candidates undergoing clinical studies, all of which are based on non-replicating viral vectors.

Ad5-nCoV-IH is an aerosol inhalation version of the Ad5-nCoV vaccine, a replication-defective adenovirus type 5 vector vaccine encoding the SARS-CoV-2 spike protein developed by Consino Biological and the Beijing Institute of Biotechnology. The Ad5-nCoV vaccine has been authorized for use in ten countries, including China and Mexico, and has demonstrated good safety and efficacy. In a study by Xu et al., the immunogenicity and safety of this nebulized inhalation vaccine were demonstrated in rhesus macaques [362]. The initial results of the phase I clinical test of the vaccine at Zhongnan Hospital (Wuhan, China) also showed that this nebulized inhaled Ad5-nCoV vaccine induced antibodies and cellular immune responses using a nebulized dose equal to one-fifth of the conventional injectable dose without serious adverse effects [342]. A phase IV clinical trial of the recombinant COVID-19 vaccine (adenovirus type 5 vector) for inhalation (Ad5-nCoV-IH) is currently underway (NCT05303584).

Another inhaled COVID-19 vaccine candidate is MVA-SARS-2-ST, developed by the German Center for Infection Research in Hamburg. It is a vector vaccine candidate based on the MVA virus, which was originally attenuated by serial tissue culture passaging of the vaccinia virus strain Ankara [363]. This resulted in the genomic deletion, which prevented the vector from replicating in human cells [364]. The vector expresses a stabilized SARS-CoV-2 S protein. A phase I clinical study is currently underway to evaluate the immunogenicity and safety of the vaccine as an inhaled booster for adults (NCT05226390).

The inhaled vaccines against SARS-CoV-2 that hold promise in entering the market are Ad5-triCoV/Mac or ChAd-triCoV/Mac. These experimental adenovirus-based vaccines were developed by McMaster University in Canada for administration as aerosol and have been approved for phase I clinical trial (NCT05094609). The vaccines are based on adenoviral vectors of human (Tri:HuAd) or chimpanzee (Tri:ChAd) origin and express three SARS-CoV-2 antigens (spike-in protein 1, full-length nucleocapsid protein, and truncated polymerase), and are designed to target the original strain of SARS-CoV-2 and variants of concern [365]. Afkhami et al. have demonstrated in murine models that this trivalent ChAd-vectored vaccine delivered via the respiratory mucosal route provided protection against both ancestral and variant strains of SARS-CoV-2, making it a potential strategy for the next-generation COVID-19 vaccine [365].

5. Summary and perspectives

The COVID-19 pandemic has drawn significant attention to the field of vaccines and has driven the fast development of vaccines, with 172 clinical and numerous preclinical COVID-19 vaccine candidates currently under investigation. In response to the challenge of widespread global vaccination and the demand for booster shots, an alternative route of immunization holds promise. Respiratory immunization presents a promising strategy against the respiratory pathogen SARS-CoV-2. This approach offers several benefits, such as delivering antigens directly to the site of pathogen invasion and infection, inducing both systemic and mucosal immunity, and mitigating the risk of infection and transmission of the virus. Currently, there are ten COVID-19 vaccine candidates in clinical trials for respiratory immunization, which primarily include viral vector vaccines and live attenuated vaccines.

However, the development of respiratory vaccines faces several challenges. Firstly, the structure of the respiratory tract imposes physiological barriers to vaccine efficacy, including the physical barrier formed by tight junctions of epithelial cells, the mucociliary clearance system, the presence of antimicrobial proteins and enzymes in the respiratory tract, and the alveolar macrophage clearance. This review has discussed the key factors that need to be considered in the formulation of respiratory vaccines in order to overcome these barriers, including the incorporation of excipients such as mucoadhesives, permeation enhancers, and enzyme inhibitors in the formulation, and the use of polymeric and lipid-based nanoparticles as delivery systems. The second challenge in developing respiratory vaccines involves the ability to produce effective and long-lasting immunity, while overcoming mucosal tolerance barriers. Sterilizing immunity is the desired goal of vaccination, although few vaccines have been able to achieve this outcome. This is due not only to the design of the vaccine itself, but also to the constant variations of the virus and the numerous and frequent exposures. Although studies in mice have demonstrated sterilizing immunity following intranasal immunization with a COVID-19 vaccine, achieving sterilizing immunity in humans through respiratory vaccines remains a challenge. Nevertheless, various mucosal adjuvants may be considered for use to enhance the efficacy of the vaccine. In addition, appropriate delivery devices are essential for the successful implementation of respiratory vaccines. The devices should be able to effectively dispense the vaccine into particles of respirable size range, while also being cost-effective and disposable for mass vaccination programs to reduce the risk of transmission of contagious diseases associated with multiple uses. More importantly, the devices should be user-friendly for the target population. Furthermore, there are several other critical issues. For example, it is required to elucidate the precise mechanisms through which vaccines stimulate mucosal and systemic immune responses. Another potential problem for the effective implementation of inhalation vaccines is the lack of representative in vivo models. There is also concern that excipients and adjuvants used in the vaccine formulation may be allergenic or irritating, potentially causing inflammation or exacerbating respiratory diseases such as allergic asthma and bronchitis. The toxicity of adjuvants and excipients must also be taken into consideration. In the past, toxin-based adjuvants (LT) have raised safety concerns for vaccines. To address this issue, new, safer, and more effective mucosal adjuvants, such as detoxification derivatives, agonists of PRRs, and cytokine adjuvants, are currently under development.

In conclusion, the development of vaccines delivered via the respiratory tract is challenging but holds great potential in the fight against COVID-19 and other respiratory infectious diseases. With continued research and innovation, it is believed that widespread market access for respiratory vaccines will become a reality in the near future.

Author contributions

X. Sun and X. He proposed the topic of the article. X. He performed the literature research and wrote the manuscript. X. Sun, G. Du, X. Chen and H. Wang revised the manuscript. All of the authors have read and approved the final manuscript.

Declaration of Competing Interest

There are no conflicts of interest to declare.

Data availability

Data will be made available on request.