Abstract
Vaccination is an effective way to prevent influenza virus infection. Currently, intramuscular vaccines are the most commonly used and can provide strong humoral immunity, but they may not induce the mucosal immune response well. A variety of pathogens gain access to the host via the respiratory tract, and the mucosa serves as the initial line of defense against bacterial invasions. Therefore, developing mucosal vaccines is a valuable strategy for preventing respiratory infectious diseases. The mucosal barrier hinders antigen delivery and immune activation, making efficient mucosal adjuvants crucial for vaccine advancement, though their use faces several obstacles. The main challenges faced by mucosal adjuvants are mucosal tolerance, delivery efficiency, and immune response balance. Future mucosal adjuvants will continue to focus on multitarget synergistic design and combination adjuvant application. The safety and efficacy of future influenza vaccines are contingent upon the judicious selection of suitable mucosal adjuvants. The creation of next-generation influenza vaccines will be made easier as our knowledge of adjuvants grows. In this review, we summarize the current progress and applications of mucosal adjuvants for influenza vaccines, with implications for the development of novel influenza vaccines and vaccines against other infectious diseases.
Introduction
Influenza viruses pose a constant threat to humans, poultry, and wild birds. Based on their nucleoproteins (NPs) and matrix proteins, influenza viruses are currently classified into four main types (A, B, C, and D) (Liang, 2023; Miyata et al., 2016). The main epidemics are seasonal outbreaks of influenza A viruses and influenza B viruses, while influenza C viruses are disseminated infections (Donatelli et al., 2017). The newly identified influenza D virus is predominantly diseased and endemic in cattle (Ruiz et al., 2022). Influenza viruses consist of eight gene segments (PB2, PB1, PA, HA, NP, NA, M, and NS) and belong to the Orthomyxoviridae family (Chen et al., 2015), which encodes at least 16 proteins (Carter and Iqbal, 2024). The various subtypes of the influenza virus are distinguished by differences in their HA and NA proteins (Bakre et al., 2021), encompassing 18 HA and 11 NA subtypes (Tong et al., 2012), and the emergence of novel influenza viruses is contingent upon the co-evolution of different HA and NA subtypes. Epidemiological surveillance of influenza viruses is important for predicting the next influenza outbreak.
Poultry and some wild birds are natural hosts of influenza A viruses, and the influenza virus is subject to constant antigenic drift and transfer within these populations (Dong and Wang, 2022), gradually evolving dominant strains through genetic mutations and subsequently evolving into seasonal pandemics through transmission. Swine act as intermediate hosts for both human influenza and avian influenza viruses (Krueger and Gray, 2013), which can cause multiple recombinations of the influenza virus. For example, a triple-rearranged recombinant H3N2 influenza strain spread in swine populations and is a recombination of the HA gene from H3N2, the NA gene from an endemic N2 strain, and the pdmH1N1 gene (Krog et al., 2017), complicating the evolution of influenza A viruses, and seasonal influenza outbreaks can occur at any time (Yoon et al., 2014). Pandemics have characterized influenza outbreaks; the 20th century saw three notable instances of pandemic influenza: the 1918 H1N1 pandemic, the 1952 H2N2 pandemic, and the 1968 H3N2 pandemic (Guan et al., 2010; Lee and Treanor, 2016; Taubenberger and Morens, 2020).
Vaccination is widely regarded as a crucial strategy in the prevention of influenza virus and pandemic propagation (Li et al., 2022). The current vaccine design strategy has moved from traditional intramuscular (IM) immunization to mucosal inoculation (Pilapitiya et al., 2023). This review describes the role and function of the mucosal immune system, presents an overview of mucosal vaccine design concepts (Song et al., 2024), summarizes adjuvants used to boost mucosal vaccines, and provides perspective on the opportunities and challenges in mucosal vaccine development.
Role and Function of Mucosa
Specialized membrane structures made of connective and epithelial tissues are called mucous membranes. Mucous membranes are the first line of protection against infections and other foreign substances because of their unique structural characteristics (Johnson et al., 2020; Li et al., 2020). The mucosal surfaces cover 400 m2 of the body (Correa et al., 2022). Given the relatively large surface area of the mucous membranes and their prolonged exposure to the external environment, maintaining a dynamic, stable, and secure immune state of the mucous membranes is an effective strategy for preventing the invasion of influenza viruses and other pathogens (Kiyono and Azegami, 2015). At the same time, mucous membranes play an integral role in the physiological processes of ingestion and inhalation.
Except for a certain number of immune cells, the epithelial parts of the mucosal surfaces of the gastrointestinal tract, respiratory tract, and genital tract also contain a certain number of other cells of the innate immune system, such as neutrophils, macrophages, dendritic cells (DCs), natural killer cells, and mast cells (M cells). These cells are the lymphoid tissue of each gastrointestinal tract, respiratory tract, and genital tract, which is also called mucosa-related lymphoid tissue (MALT), and account for a large proportion of the MALT (Chen, 2000). The first target after mucosal vaccination is the mucosal surface. The surface of the mucosa often contains M cells, which uptake and present antigens. These cells facilitate the absorption of antigens from the surrounding environment and transport them to the underlying lymphoid tissues (Chen, 2000). Upon entering the MALT, the antigen is quickly taken in and processed by antigen-presenting cells (APCs), such as subepithelial DCs and macrophages. Subsequently, these cells pass on the processed antigen to B and T cells in the MALT. After antigen sensitization, B cells undergo proliferation and transform into IgA-binding cells. Subsequently, these B cells exit the MALT and enter the systemic circulation, eventually reaching various mucosal sites, including the original site for differentiation into sIgA-producing plasma cells (Fig. 1).
Adjuvants promote antibody production. Pattern recognition receptors on antigen-presenting cells (APCs) may be impacted by mucosal adjuvants. Certain adjuvants can selectively act on certain receptors on APCs by imitating the size and form of pathogens. This causes the release of related inflammatory factors, like IL-6 and IFN-γ. These factors increase the absorption of antigens, trigger both innate and adaptive immune responses, help effector T and B cells mature and activate, turn them into memory B cells, and stimulate effector plasma cells to produce more antibodies. Adjuvants can therefore be included in vaccinations to encourage the formation of antibodies (By Figdraw).
Mucosal Immune System
Mucous membranes serve as a connection between the internal and external environments of an organism, and most pathogens gain access through mucosal surfaces. Mucosal vaccination is an effective strategy for preventing and controlling influenza viral infections. It can enhance the mucosal immune response and lower viral transmissibility and pathogenicity (Bernasconi et al., 2016; Correa et al., 2022). There are two kinds of sites in the mucosal immunity: induction sites and effector sites. The main function of the induction site is the capture and presentation of the antigen and the activation of cells and other functions. However, the main function of the effector site is responding to the ingested antigen. The circulation of cells between the induction site and the effector site is an important function of mucosal immunity. The mucosal immune system communicates between the induction site and the effector site mainly through lymphocyte homing; that is, immune cells sensitized in the induction site enter the blood circulation and gradually differentiate and mature (Li et al., 2020).
The mucosal immune system protects the body from the invasion of foreign bodies (Li et al., 2013). Therefore, for the sake of the health of the body, we should maintain the mucosal immune system in a safe and stable state. The mucosal immune system contacts the external environment of the organism and may be invaded by pathogens at any time. Once the pathogenic microbes have invaded, mucosal immunity can play a timely role in recognizing and removing pathogens that have invaded the mucosal surfaces, thus preventing the spread of pathogenic microbes in the body. Additionally, this system rigorously regulates the exchange of both beneficial and harmful substances within the organism and preserves the dynamic balance between the organism’s internal and exterior surroundings (Holmgren and Czerkinsky, 2005).
Furthermore, the mucosal immune system differs from the systemic immune system in certain ways. While systemic immunity does not engage the mucosal immune system and usually only generates systemic responses, mucosal immunity usually elicits both local mucosal and whole-body systemic immunological responses. Designing mucosal adjuvants that work requires an understanding of these pathways.
Adjuvant Mechanisms
Adjuvants are necessary to overcome the mucosal immune system’s innate tolerance and boost antigen-specific responses because of its special characteristics. Adjuvants are described as additives in vaccines (Fan et al., 2023) that stimulate and improve the strength and duration of the immune response (Reed et al., 2013). These additives can range from a variety of small-molecule chemicals to numerous complex extracts of natural substances (McKee et al., 2007). Adjuvants fall into two categories: delivery mechanisms and immunostimulants (Yu et al., 2024; Zhao et al., 2023). The potential benefit of mucosal immunization over conventional immunization is the triggering of immune defenses in mucosal and systemic tissues, preventing pathogens from invading mucosal surfaces and creating a protective layer in the body’s first line of defense (Li et al., 2020).
Currently, there are limited adjuvants available for mucosal vaccines in clinical practice. To produce a strong immune response against infections, an efficient delivery system or a potent mucosal adjuvant must be used (Gao and Guo, 2023). Effective mucosal adjuvants enhance immune responses by promoting prolonged antigen release. They target APCs, which is essential for creating vaccines that work by focusing the immune response on Th1 or Th2 pathways (Newsted et al., 2015).
Application in Influenza Vaccines
Pathogens typically interact with the host’s mucosal surfaces and penetrate mucosal tissues by secreting harmful chemicals, resulting in systemic or organ-specific illnesses (De Magistris, 2006). It is therefore vital to stimulate mucosal defenses to maintain the body in a state of heightened defense. Mucosal vaccines effectively stimulate robust mucosal immune responses, producing high levels of sIgA antibodies, unlike non-mucosal inoculation, which results in slower and lower antibody yields.
Mucosal vaccination has many advantages over other vaccination approaches, which can largely be attributed to the nature of the immune response induced by the mucosal route. In the early stages of an infection, antigen-specific IgA antibodies are produced at the site of infection through mucosal immunization (De Magistris, 2006). This pathogen-specific local response is of relevance not only for the prevention of infectious disease in the vaccinated person but also for the clearance of the healthy carrier state and its induced spreading to unprotected individuals (De Magistris, 2006). Second, mucosal-primed lymphocytes express mucosal-specific homing receptors, which means that a specific response can be induced at a distant site through immunization at one mucosal site. Third, mucosal vaccination can induce a mucosal immune response. Furthermore, it is also possible to use mucosal vaccination to target other types of pathogens such as non-mucosal pathogens that are not located in the blood or on the skin (De Magistris, 2006).
Mucosal vaccines also have several significant practical benefits over other vaccines. First, the need for needle injection is eliminated, which means that the vaccine is less painful. Second, it is easy to administer mucosal vaccines and does not require professionals . Furthermore, fewer adverse reactions and booster vaccinations occur in comparison with injected vaccines (De Magistris, 2006). In Table 1, we summarize some mucosal adjuvants used in influenza vaccines. The selection of appropriate mucosal adjuvants is key to exerting an efficient mucosal immune response and supports the initiation of the transition from innate to adaptive immunity (Wong et al., 2014). Of course, different adjuvants have different characteristics. When using adjuvants, one should fully understand their properties. For example, regarding safety, sustained-release effects, and biodegradability, adjuvants should be used appropriately based on their effects. Table 2 summarizes a comparison of some common adjuvants.
Mucosal Adjuvants for Influenza Vaccines
Characteristics and Prospects of Mucosal Adjuvant Platforms for Influenza
Antigen-Coupled Adjuvants
Transferrin receptor protein 1 (TFRC), also known as transferrin receptor/TfR, is a type II transmembrane glycoprotein that interacts with transferrin (Tf) and is essential for cellular iron uptake. This is achieved by interacting with iron-binding Tf (Mann et al., 2016). Oral vaccination has several advantages over traditional parenteral and other routes of vaccination, and improved ways of vaccination can sometimes boost the efficiency of vaccine immunization. Tf was examined by researchers to see if it might be used as a conjugate vaccine method that could target carriers and penetrate mucosal tissues (Mann et al., 2016). High amounts of antigen-specific serum IgG were found when influenza virus HA and Tf were coupled as a vaccination and administered sublingually. The findings demonstrated that Tf can improve vaccination immunogenicity by acting as a combination mucosal adjuvant (Mann et al., 2016).
Lactobacillus is commonly used by researchers as an oral vaccination carrier (Lei et al., 2015). The cholera toxin B (CTB) subunit as a mucosal adjuvant improves the immunogenicity of Lactococcus lactis-vectored vaccines used in combination with it. In order to create a L. lactis-vector vaccine, the NP gene of the A/California/04/2009 (H1N1) influenza virus was subcloned into the L. lactis expression vector pNZ8008. Oral injection of L. lactis/pNZ8008-NP in conjunction with the mucosal adjuvant CTB component was used to assess the immunogenicity of the combination in mice (Lei et al., 2015). The scientists came to the conclusion that oral immunization with 5 × 1011 CFU of L. lactis/pNZ8008-NP plus 1 µg of CTB adjuvant produced notable immune responses and may provide cross-protective immunity against several influenza A viruses (Lei et al., 2015).
Bacterium-Like Particles
Bacterium-like particles (BLPs) are regarded as a unique vaccine delivery carrier because of their benefits, which include high safety, good stability, great carrying capacity, and high mucosal transport efficiency. However, there is still much to learn about the use of BLPs to improve vaccine efficacy (Zhou et al., 2023).
Using the right immunostimulatory substances or adjuvants is essential to the effectiveness of intranasal (IN) vaccination (Van Braeckel-Budimir et al., 2013). Numerous adjuvants have been found to be viable options for mucosal vaccinations. Despite the high immunogenicity of many adjuvants, safety and regulatory issues frequently restrict their clinical application (Van Braeckel-Budimir et al., 2013). BLP is a perfect adjuvant for nasal influenza vaccines to boost their immune-stimulating effects. Natalija et al. administered FluGEM-A, a product that contains BLPs and a seasonal subunit vaccination, intranasally to mice (Van Braeckel-Budimir et al., 2013). According to the findings, animals given FluGEM-A intranasally had considerably higher antibody titers than mice given adjuvant-free subunit vaccinations in the same manner. When administered intranasally, BLP functioned as an adjuvant for the inactivated influenza vaccine. The study also demonstrated that BLP can elicit robust systemic and mucosal B cell immune responses (Keijzer et al., 2014).
The effects of IM and IN inoculation with inactivated influenza vaccines enhanced with bacterial-like particles were compared by Aalzen et al. (de Haan et al., 2012). The results show that compared with traditional IM injections, IN vaccination can enhance systemic and local antibody responses, thereby providing better protection against both homologous and heterologous influenza viruses. The development of mucosal adjuvants is a new approach toward the design of next-generation vaccines (e.g., BLPs) (Van Braeckel-Budimir et al., 2013).
CpG-ODN Adjuvant
TLR9 can specifically identify the CpG DNA that pathogens release when they infiltrate the body, which triggers an immune response. The TLR9-MyD88 complex is formed when TLR9’s intracellular TIR domain enlists myeloid differentiation factor 88 (MyD88) following its recognition of its ligand. Tumor necrosis factor receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinases (IRAKs) are two more downstream signaling molecules that are activated by this complex (Fig. 2) (Kayraklioglu et al., 2021). CpG oligodeoxynucleotides (ODNs) can activate immune cells and stimulate the production of specific cytokines. As immunomodulators, they can specifically stimulate the expression of Toll-like receptor 9 (TLR9). TLR9 expressed on human plasma cells and DCs is responsible for inducing innate immune responses characterized by the production of Th1 cells and pro-inflammatory cytokines (Kayraklioglu et al., 2021).
Mucosal adjuvant-related TLR/STING/IL signaling pathways. TLR9 uses the MyD88-dependent pathway to send downstream signals. Pro-inflammatory cytokines and interferons are produced when the adaptor proteins MyD88 and IRAKs trigger the NF-κB and IRF signaling pathways. The cGAS can identify pathogen DNA and then catalyze the production of 2′,3′-cGAMP. 2′,3′-cGAMP translocates along with its “closed” conformation binding to STING. Type I interferons, pro-inflammatory cytokines, and chemokines are all upregulated when transcription factors IRF3 and NF-κB are phosphorylated. The STING signaling pathway can also be triggered by pathogen-derived c-di-GMP, c-di-AMP, and 3-cGAMP. By interacting with the ligand myeloid differentiation primary response protein 88 (MyD88) and activating the TNF receptor-associated factor 6 (TRAF6) and mitogen-activated protein kinase (MAPK) signaling modules, as well as by binding to it and activating nuclear factor-κB (NF-κB), IL-18/IL-1β produces and releases a variety of cytokines and chemokines (By Figdraw).
Transmission of low-pathogenicity avian influenza (LPA) H9N2 influenza virus (H9N2 WIV) severely increases the risk of a new influenza pandemic, and the mucosal barrier is a major impediment to vaccine development. CpG-ODNs are acknowledged as adjuvants that can target DCs downstream and have been shown to be successful in boosting systemic and mucosal immune responses. According to Tao et al.’s in vitro and in vivo tests, CpG prevents H9N2 WIV from spreading further (Qin et al., 2015). Trans-epithelial dendrites (TEDs), which are employed to capture the virus, are formed when DCs are recruited to nasal epithelial cells (ECs). The chemokine CCL20, which is released by nasal ECs, is a major inducer of the recruitment of DCs and the development of tertiary lymphoid structures. In order to deliver antigens, DCs can swiftly recruit viruses and go to cervical lymph nodes (CLNs). The results of the study indicate that CpGs can promote H9N2 WIV trafficking via TEDs in nasal DCs, which may be a unique strategy to enhance optimal adaptive immune responses (Qin et al., 2015). Additionally, they demonstrated that the CpG adjuvant promotes an effective antigen-specific immune response and makes it easier for the intestinal epithelial barrier to absorb the H9N2 complete inactivated virus vaccine (Yin et al., 2015).
Some of the delivery system-related factors that influence a vaccine’s immunogenicity include its slow-release effect at the injection site, the strength of its immunostimulatory properties, and the characteristics of its antigen stability (Zhao et al., 2014). Singh et al. examined how encapsulated and nonencapsulated PLGA nanoparticles affected the immune response to the inactivated H9N2 influenza virus (Singh et al., 2016). The findings demonstrated that administering unencapsulated CpG ODN 2007 intramuscularly in combination with an inactivated influenza A virus vaccination produced higher antibody titers than encapsulated CpG ODN 2007. Furthermore, compared with unencapsulated CpG ODN 2007, inactivated CpG ODN 2007 encapsulated in avian influenza virus vaccinations administered via the aerosol method produced a noticeably stronger mucosal immune response. CpG ODN 2007 can act as an efficacious mucosal adjuvant (Singh et al., 2016).
Cytokine Adjuvants
Cytokines are involved in regulating cell–cell interactions. They are the main stimulatory molecules of T cells and B cells, which are indispensable in the process of immune response. Cytokines as adjuvants mainly affect Th1/Th2 cell immunity (Lazear et al., 2019). Cytokines, such as interferon (IFN), tumor necrosis factor-α (TNF-α), interleukin (IL), and colony-stimulating factor (CSF), are secreted by immune cells and play a key role in regulating the immune system (Opal and DePalo, 2000).
In 1957, IFN was first discovered. When a virus infects cells in vitro, they produce this substance, and scientists have studied its characteristics (Balkwill, 1989). IFN is a naturally occurring protein that has several biological functions, including regulating cell division and directing the immune system’s antiviral and immunoregulatory responses. This is accomplished by stimulating the transcription of several useful genes within the cells (Liu et al., 2019). This influences how different signaling pathways work in concert. The possibility of using recombinant porcine interferon (PoIFNα) as an adjuvant was investigated. At 6 weeks of age, piglets received an influenza vaccine that contained PoIFNα in their nasal canals. The PoIFNα vaccine group increased the levels of virus-specific antibodies, according to the results (Liu et al., 2019).
Adjuvants for mucosal vaccinations can be cytokines from the interleukin-1 (IL-1) family, according to several research (Kayamuro et al., 2010). When IL acts as an adjuvant, the NLRP3 inflammasome can quickly recognize pathogens and activate caspase-1 to cleave the precursors of IL-1β and IL-18. IL-1β recruits neutrophils to the site of mucosal infection, accelerating pathogen clearance, while IL-18 promotes EC repair and enhances NK cell residency in the mucosa (Fig. 2) (Pang et al., 2013). Therefore, considering the above, many researchers evaluated the effectiveness of recombinant adenoviral vector vaccines with rAd-IL-1β or rAd-IL-18 vaccines for nasal immunization in BALB/c mice (Lapuente et al., 2018). Co-expression of IL-1β and antigen has been shown to activate chemokine and adhesion molecule expression, DC recruitment, and local memory T cell generation. These findings validate the significance of this study for vaccine development, particularly given the role played by tissue-resident memory (TRM) cells in mucosal immunity (Lapuente et al., 2018).
TNF-α was fortuitously identified in a serendipitous discovery made during a study in which BCG-vaccinated mice were injected with bacterial lipopolysaccharides. The isolation of the serum revealed the presence of a substance capable of causing hemorrhagic necrosis of tumors, which was subsequently defined as TNF (Balkwill, 2009; Jaco et al., 2017). TNF-α has been demonstrated to possess adjuvant properties against viral infections in a variety of model systems (Brunner et al., 2000; Chen et al., 2002). During a viral infection, TNF-α affects immune responses, including the activation of the immune system, the maturation of DCs, and the clearance of pathogens (Calzascia et al., 2007; Kayamuro et al., 2009). One study has shown that mutant TNF-α lacking lysine residues has higher biological activity than wild-type TNF-α (wTNF-α) (Shibata et al., 2004). Kayamuro et al. mixed influenza virus antigen with mTNF-K90R to immunize mice to test its effect (Kayamuro et al., 2009). The results demonstrated that mTNF-K90R enhances systemic and mucosal immune responses, suggesting its potential to serve as an effective mucosal vaccine adjuvant (Kayamuro et al., 2009).
Cyclic GMP-AMP (cGAMP) is an intracellular second messenger that can directly activate the cGAS-STING pathway. For specific targeting of mucosa, adjuvants are often combined with cationic polymers to penetrate the epithelial layer and target mucosal DCs, initiating a signaling cascade by activating stimulator of interferon genes (STING) to produce type I IFNs and other immune mediators (Fig. 2) (Yuan et al., 2024). Researchers immunized mice intranasally with influenza virus HA vaccine alone and HA vaccine plus cGAMP adjuvant (Takaki et al., 2017), and the results showed that cGAMP enhanced IgA and IgG antibody production, activated nasal mucosa-associated lymphoid tissue (NALT) and immune cells throughout the body, and created an environment conducive to antibody production (Takaki et al., 2017).
Combined Adjuvant
It has been demonstrated that influenza virus vaccines are ineffective in inducing immune-protective effects in organisms at high risk of infection. Additionally, organisms infected with influenza viruses have been observed to activate a diverse range of pattern recognition receptors (Wong et al., 2021). Novel adjuvants that can simultaneously activate several receptors must be discovered in order to improve immune protection. They will more closely resemble the normal immune system, which will enable them to generate adaptive antibody responses and stronger antiviral cellular immunity (Wong et al., 2021). Developing successful vaccines requires a multifaceted strategy. Carefully selecting the optimum adjuvants, antigens, and delivery techniques in the right formulation is crucial to producing vaccines that are more immunogenic, safe, and stable (Kaurav et al., 2018; O’Hagan and Valiante, 2003). This makes it possible for vaccinations to produce a thorough and effective immunization.
Wong et al. developed a novel vaccine formulation by combining two adjuvants with distinct biological properties: an oil-in-water nanoemulsion (NE) and an RNA-based RIG-I agonist. They discovered that the NE balanced the Th1/Th2/Th17 response by stimulating antigen apoptosis and activating Toll-like receptors (Wong et al., 2021). Th1 polarization and IFN-Is were subsequently triggered by the RIG-I agonist. In addition to demonstrating positive immunological effects, the combination of these two adjuvants in vaccination applications further confirms the significant utility of combined adjuvants in the application of influenza virus vaccines and opens up new avenues for vaccine research and use (Wong et al., 2021).
The unique strain of avian influenza known as H7N9 was first discovered in eastern China in March 2013. Rapid progression to pneumonia, respiratory failure, and acute respiratory distress syndrome (ARDS), which eventually results in mortality, are the disease’s hallmarks (Li et al., 2014). In order to suppress the H7N9 influenza outbreak, Xu et al. assessed the immune response of recombinant human interleukin-2 (JY) and chitosan (CS) as adjuvants in conjunction with inactivated split vaccines by nasal vaccination against the H7N9 influenza A virus (A/Shanghai/02/2013) (Xu et al., 2018). The results showed that the adjuvant-induced IgG and sIgA titers were much greater than those in the IM injection group, and the mice vaccinated with the JY adjuvant showed significantly better hemagglutination inhibition (HI) than the non-adjuvanted group. The JY adjuvant has been demonstrated to provide both systemic humoral response and local mucosal immunity when used in conjunction with H7N9 inactivated nasal spray immunization. This could strengthen immunity against the respiratory transmission of the H7N9 influenza virus (Xu et al., 2018).
Inorganic Adjuvants
Over the past 10 years, inorganic adjuvants have made tremendous progress in the fields of nanostructure production, structural modulation, and functional design. Generally speaking, inorganic adjuvants aid in prolonged antigen release, improve immunogenicity, effectively distribute antigens, and elicit a robust immune response (Li et al., 2018).
When paired with a cell-penetrating peptide-linked polymer, Ukawa et al. have shown that poly N-vinyl acetamide-co-acrylic acid modified with doxorubicin has the potential to function as an effective mucosal vaccination (Ukawa et al., 2019). Subsequently, the researchers endeavored to develop cell-penetrating peptides with biodegradability for potential clinical applications (Ukawa et al., 2019). Hyaluronic acid was synthesized by modifying glucuronic acid with tetraglycine-
Alkyl glycoside (APG) is a new type of highly effective, nontoxic, and biodegradable nonionic surfactant formed by the catalytic dehydration of the hemiacetal hydroxyl group of glucose and the hydroxyl group of fatty alcohols (Wu et al., 2017). The compound can be employed as an adjuvant for nasal mucosal vaccines due to its capacity to enhance the uptake of macromolecules by the nasal mucosa. Gellan gum is a biopolymer made in a 2:1:1 ratio of glucose, glucuronic acid, and rhamnose. When exposed to cations (Na+, K+, and Ca2+), it can neutralize and react with them due to its acidic characteristics. When used in vaccine applications, this leads to a solution-to-gel phase shift that can increase the viscosity of vaccines (Wu et al., 2017). When it comes to mucosal vaccinations, it is possible to achieve both the optimization of vaccine efficacy over an extended period of time and the extension of vaccine retention at the mucosa (Gomes et al., 2023; Villarreal-Otalvaro and Coburn, 2023). Among natural polymers, CS is an uncommon alkaline polysaccharide. It is frequently utilized in biomedicine and preparations due to its biodegradability, low toxicity, and strong biocompatibility. CS has been shown to be a novel nano-drug carrier, and one of its key characteristics is its unique mucosal adherence (Collado-González and Esteban, 2022; Wang et al., 2020; Wu et al., 2017).
When administering a split influenza vaccination intranasally to BALB/c mice, the researchers examined the efficacy of three possible mucosal adjuvants (APG, colistin, and CS). APG was found to be the most immunologically effective IN adjuvant in a comparison of immunization with the three adjuvants; mice utilizing APG had lower virus titers, more entire lung sections, and the least amount of weight loss and survival following challenge in all adjuvant groups (Wu et al., 2017). These findings have shown that APG improves mucosal and systemic immune responses. Additionally, APG has been demonstrated to be a mucosal adjuvant in the context of influenza vaccinations (Wu et al., 2017).
Particulate Antigen Carrier
Chitosan nanoparticles and derivatives
An attractive delivery method for IN vaccinations is CS. CS has been widely used as a carrier for peptide, protein, and DNA vaccines due to its nontoxicity, advantageous bioadhesion, and biodegradability (Chua et al., 2012; Sawaengsak et al., 2014; Yang et al., 2009). In order to overcome the insolubility and aggregation problems that impede its use, researchers have chemically and physically altered CS (Singh et al., 2018). CS has been extensively studied in relation to immunological targeting, biosafety, and the mucosal barrier. It is expected that in the future it will be developed into a mucosal adjuvant with many biological activities (Qin et al., 2020).
Due to its inherent qualities, the material known as “CS” has been used more frequently in recent decades to deliver a range of medications (Amidi et al., 2007; Chua et al., 2012; Sawaengsak et al., 2014; van der Lubben et al., 2001). CS has been shown to improve immune function and is a desirable nasal vaccine delivery method (Bae et al., 2016). The substance’s adhesive qualities allow it to evade the mucociliary clearance process, which prolongs the residence time of vaccine components in the nasal cavity (Bae et al., 2016), improving the immunogenicity and effectiveness of vaccines (Alpar et al., 2005). A technique for creating CS nanoparticles employing ionic cross-linking with sodium tripolyphosphate (TPP) at a 1:0.6 ratio and stirring for 2 h was devised by Sawaengsak et al. (2014). When a split influenza HA vaccine encapsulated in CS/TPP nanoparticles was administered intranasally, systemic and mucosal antibody levels were considerably higher than when a split influenza HA vaccine was administered alone (Sawaengsak et al., 2014). The high number of IFN-gamma-producing cells in the spleen showed that the nanoparticle-encapsulated vaccination also triggered a cellular immune response.
Additionally, the CS nanoparticle-encapsulated split influenza HA vaccine completely protected the inoculated mice and markedly reduced influenza-related morbidity (Sawaengsak et al., 2014).
Because of their controlled manufacturing and structural rigidity, inorganic nanoparticles have been used as adjuvants and antigen delivery vehicles (Kheirollahpour et al., 2020; Liu et al., 2021). Iron oxide enzyme (IONzyme) has been shown to exhibit a range of enzymatic activities, including those associated with peroxidase, catalase, and lipoxygenase-like enzymes (Qin et al., 2019). At neutral pH, IONzyme helps scavenge reactive oxygen species (ROS) with catalase-like action. However, in acidic pH settings, IONzyme exhibits peroxidase-like activity, which speeds up the production of ROS. IONzyme is transported to DCs in the mucosal immune system for processing. Therefore, the age of the DCs is a major factor in the effectiveness of later immunization (Qin et al., 2020). A successful model of a CS-functionalized IONzyme-based nasal mucosal delivery vaccine for H1N1 influenza viruses was presented by Tao et al. It is extremely protective against the deadly threat of influenza viruses (Qin et al., 2020). It was anticipated that CS modification would lead to an increase in peroxidase-like activity and the catalysis of ROS-dependent DC maturation in addition to a notable improvement in the antigenic adhesion of nasal mucosa. This finding opens up new research directions for novel mucosal vaccinations.
Liposome nanoparticles
Nucleic acids are transported by liposome (LS) nanoparticles into cells. It is made up of PEG lipids, cholesterol, neutral auxiliary phospholipids, and ionizable cationic lipids (Mitchell et al., 2021). This indicates that a variety of substances, including hydrophilic, hydrophobic, and lipophilic ones, are transported by liposomes (Mitchell et al., 2021; Sarfraz et al., 2018). LS is a very promising family of nanoparticles with special characteristics such as post-synthesis modification, high specific surface area, and variable nanosize, as adjuvants for vaccines, which could convert antigens with low immunogenicity into antigens with high immunogenicity (Krasnopolsky and Pylypenko, 2022), inducing long-term specific effects of antibodies, and presenting both hydrophilic and hydrophobic antigens (Krasnopolsky and Pylypenko, 2022). Experimental results indicated that LS, when utilized as a vaccine adjuvant, has the capacity to enhance immune responses. In addition, it has been reported that it is biodegradable and automatically eliminated from the organism, so it meets the requisite safety criteria for an adjuvant. The efficacy and safety of LS as a carrier for antigen delivery systems have been demonstrated in clinical studies of anti-hepatitis A, influenza, and COVID-19 vaccines (Krasnopolsky and Pylypenko, 2022).
Santosh et al. engineered a subunit vaccine consisting of liposomal nanoparticles carrying 10 highly conserved B and T cell epitope peptides (Dhakal et al., 2018; Sia et al., 2021). The researchers vaccinated pigs intranasally to boost their immunity against the H1N1 swine influenza virus (SwIAV). Several assays showed that the liposomal peptide influenza vaccine improved HI antibody titers and increased sIgA antibody production. Meanwhile, IN immunizations improved protection against the mutant strains, increased T helper/memory cell frequencies, enhanced mucosal immune responses, and reduced viral loads in the nasal mucosa and lungs (Dhakal et al., 2018; Sia et al., 2021).
LS have been demonstrated to function effectively as adjuvants, offering a safe and efficient means of enhancing immunization (Ma et al., 2022). They protect antigens from degradation, facilitate antigen delivery, and induce the maturation of DCs, thereby promoting an effective immune response (Qu et al., 2018). Moreover, cationic liposomes exert a more pronounced adjuvant effect than neutral or anionic liposomes (Kolašinac et al., 2021; Ma et al., 2022; Nagy et al., 2022; Tang et al., 2021). Ma et al. hypothesized that the use of cationic liposomes as immunomodulators could enhance the immunological effects of nasal mucosal immunity (Ma et al., 2022). The researchers constructed a cationic lipid, 1,2-dimyristoyl-rac-glycerol (DDA), as a carrier and adjuvant, and an immunomodulatory agent,
Qu et al. recently investigated whether cationic DDA/trehalose dibehenate (TDB) liposomes binding to influenza A viral antigen (H3N2) improved humoral and mucosal immune responses in C57BL/6 mice (Qu et al., 2018). Because of its advantages, which include high safety, good stability,enormous carrying capacity, and high mucosal transport efficiency, BLPs are considered a unique vaccine delivery carrier. But there is still a lot to learn about using BLPs to increase the effectiveness of vaccines (Qu et al., 2018). The benefits of liposome nanoparticles (LNPs) as an adjuvant are further demonstrated by this study.
TNF/CHP Nanoparticles
The application of nanoparticles made from biocompatible polymers has seen a sharp rise in interest recently (Bai et al., 2023). The main cytokine that promotes inflammation is TNF-α (Technau, 2008). It is currently thought to be a crucial part of the host’s defense against invasive pathogens, as evidenced by the stimulation of inflammatory reactions, induction of DC maturation, and activation of innate and adaptive immunological responses (Nagatomo et al., 2015; Wajant et al., 2003). A polysaccharide branching starch that has been chemically altered with cholesterol molecules is called cholesteryl hydrophobized pullulan (CHP). In an aqueous solution, it can self-assemble into nanoparticles and use hydrophobic interactions to trap and restrict other tiny molecules inside its cavity (Akiyama et al., 2007; Prajapati et al., 2013).
TNF-α was encapsulated in CHP by Daiki et al. to create TNF/CHP nanoparticles (Nagatomo et al., 2015; Nagatomo et al., 2015). The durability of the TNF/CHP storage, which stimulates the generation of both humoral IgG and mucosal IgA antibodies, was shown by the team’s inoculation of the TNF/CHP nanoparticles in a mouse animal model. Additionally, the vaccine’s protective impact against viruses was shown by viral challenge studies in mice, which also encouraged effector T cell and B cell activation and antigen uptake by DCs. Their findings serve as a basis for the later creation of novel vaccinations.
Virus-Like Particles
Viral capsid proteins self-assemble to form highly organized protein nanostructures known as virus-like particles (VLPs) (Mohan et al., 2017). These proteins resemble those present in naturally occurring viral particles. VLPs are highly immunogenic and bioactive, which makes them an effective way to administer vaccines, since they can identify receptors and induce innate immunity. Successful VLP immunizations can generate long-lasting, potent antibodies that can quickly eradicate the virus and protect the receiver against disease (Mohan et al., 2017).
In one study, researchers developed a new vaccine based on influenza virus hemagglutinin (HA) that was injected intranasally into mice using VLPs embedded with CCL28. This strategy opened up new avenues for vaccination research (Mohan et al., 2017). Their results showed that compared with the experimental group alone, the vaccine produced a significantly stronger and more persistent virus-specific antibody. The chimeric VLP (cVLP) group demonstrated robust immunoprotection when vaccinated mice were exposed to the H3N2 virus at month 8. This was demonstrated by HI titers and significantly elevated and persistently releasable IgA antibody levels. Meanwhile, inflammation and viral load declined in the cVLP group (Mohan et al., 2017). VLPs can be used as potential vaccine delivery vehicles, as this study shows.
Demetrius et al. created a VLP vaccine that contained the 1918 influenza A virus’s surface glycoproteins HA and NA and assessed the virus’s pathogenicity using the A/swine/Iowa/15/30 (H1N1) assay (Matassov et al., 2007). The findings showed that, in comparison with the control group, mice inoculated with the VLP alone had noticeably reduced virus titers in the nose and lungs. In one study, Peter et al. injected VLPs made of the hemagglutinin (HA), neuraminidase (NA), and membrane (M1) proteins of the A/Hong Kong/1073/99 (H9N2) virus subcutaneously into mice (Pushko et al., 2005). It was discovered that the resulting assembled VLPs inhibited influenza virus multiplication and induced serum-specific antibodies to the influenza A/Hong Kong/1073/99 (H9N2) virus. The potential of VLP as a vaccine delivery vehicle is confirmed by this investigation.
Clinical Translation of Mucosal Adjuvants
The primary objective of mucosal adjuvant research is to benefit humanity. Mucosal adjuvants have been shown in numerous research to enhance immune responses and facilitate vaccination procedures. In a Phase I clinical trial, researchers compared Advax-CpG with protein-based COVID-19 or influenza vaccinations either orally or sublingually. The results demonstrated that the adjuvant is safe, well tolerated, and provides strong defense against the heterologous Omicron BA.5 virus and the fatal influenza virus (ClinicalTrials.gov, 2024a). Experiments have shown that the flagellin protein adjuvant is safe and effective. Researchers carried out an early Phase I investigation to further evaluate the safety, immunogenicity, and tolerability of the recombinant flagellin protein adjuvant KFD1 in people (ClinicalTrials.gov, 2024b). The Center for Human Drug Research in the Netherlands conducted a first-in-human experiment to assess the safety, immunogenicity, and reactogenicity of a nasal seasonal influenza vaccine (FluGEM) adjuvanted with Gram-positive enhancer matrix (GEM) particles. The results demonstrated that it is safe and well tolerated, and it produced noticeably higher HI titers (van der Plas et al., 2024).
The use of nasal vaccinations is growing. A Phase I–Ib, double-blind, randomized, repeated-dose, single-center safety and immunogenicity study of nasal Poly-ICLC (Hiltonol®) in healthy COVID-19-vaccinated adults was conducted. The results of the study indicate that this adjuvant is safe, although its mucosal immune effect is weak and needs to be strengthened (ClinicalTrials.gov, 2020). Similarly, a clinical trial investigation evaluated the safety and immunogenicity of the novel NE mucosal adjuvant (W805EC) by administering it intranasally alongside the authorized seasonal influenza antigen. The findings showed that the W805EC adjuvant can produce both systemic and mucosal immunity following a single IN vaccination and is safe and well tolerated in healthy adult volunteers(Stanberry et al., 2012). Table 3 summarizes some real cases of clinical translation of mucosal adjuvants. These clinical trials offer secure and trustworthy information for mucosal adjuvant research, which can promote the improved application of mucosal adjuvants for the benefit of humanity.
Mucosal Adjuvants in Clinical Trials for Influenza and COVID-19 Vaccines
However, there are still a lot of difficulties and safety issues. Thorough monitoring of mucosal adjuvants’ systemic toxicity, cost-effectiveness, safety, and effectiveness is required. At the same time, customized formulations and focused delivery are new areas of study. The study of mucosal adjuvants may pave the way for novel vaccine development, and further progress in this area will benefit humanity.
Conclusion
The mucosal immune system, a vital component of the body’s internal environment, plays a pivotal role in resisting influenza viruses. Blocking influenza virus infection in the mucosa is one of the current and future directions of influenza virus vaccine development. Unlike conventional vaccines, mucosal influenza vaccines can induce not only humoral immunity but also mucosal immunity, which can be a good way to stop influenza viruses at the beginning of their transmission.
The effectiveness of a vaccine is impacted by several factors, such as the way the vaccine is prepared, how long it is stored, the route of administration, the use of adjuvants, and many other factors. Currently, influenza vaccines mainly include egg-based vaccines, cell-based vaccines, and recombinant influenza virus vaccines. In the meantime, in an attempt to cope with the mutation of the influenza virus and to improve the immune response of the vaccine, vaccine production is moving toward multiplex polyvalent vaccines, and the current influenza vaccine is usually trivalent or quadrivalent. Trivalent inactivated influenza vaccines are formulated to provide protection against three types of influenza viruses: influenza A H1N1, influenza A H3N2, and one influenza B virus. In contrast, tetravalent vaccines are formulated to provide broader protection against influenza A and B viruses, in addition to the three types covered by trivalent vaccines.
The current focus of research into the influenza virus vaccine is on developing methods for delivering the vaccine and the creation of mucosal adjuvants. The traditional routes of administration through subcutaneous or IM injection do not work well for vaccine efficacy, which can be improved by mucosal routes (e.g., nasal mucosa and oral mucosa). Particularly popular is vaccination through the nasal mucosa, where nasal vaccines induce not only sIgA antibodies but also serum IgG antibodies, which provide cross-protection against homologous and heterologous viruses. Adding appropriate adjuvants in mucosal vaccines is particularly important for achieving durable and effective immunity. Existing mucosal immune adjuvants include antigen-coupled adjuvants, BLPs, CpG-ODN adjuvants, cytokine adjuvants, inorganic adjuvants, particulate antigen carriers, LNPs, TNF/CHP nanoparticles, and VLPs.
Mucosal adjuvants represent a significant area in influenza vaccine development. Although some progress has been found in mucosal adjuvant development over the past decades, the precise mechanism of action of mucosal adjuvants remains unclear. This impedes our ability to comprehend the principles underlying the design and optimization of these adjuvants. It is clear that in the future, studies should try to get a deeper understanding of the complicated and multifaceted nature of the mucosal immune system and how the mucosal adjuvants interact with the immune system. The main mucosal adjuvants that have been identified so far consist of bacterial substances, cytokines, and antigen delivery systems. However, the effects of these adjuvants are not universal, and they may bring some safety problems. So, studies in the future should focus on more efficient, safe, and broad-spectrum mucosal adjuvants.
Moreover, with the development of biotechnology, such as gene editing and synthetic biology, we believe that these will provide new opportunities and approaches for novel mucosal adjuvants. In summary, the future progress of novel mucosal adjuvants will depend on our understanding of the mucosal immune system, the adaptation of various mucosal adjuvants to influenza vaccines, and novel technologies. With the great achievements of the past and present and the fast development of science and technology, we believe that more efficacious and safer mucosal adjuvants will be developed in the near future, which will provide better impetus for the development of the next-generation influenza vaccines.
Authors’ Contributions
G.W. wrote the first draft of the article. H.F. and L.W. contributed to conception and design of the review and revised the article. X.W., Y.T., S.L., C.L., Y.Y., S.X., H.X., and M.L. are involved in the integration of the related information. J.S. and Y.H. provided critical review for this article. All authors reviewed and approved the submitted version.
Footnotes
Author Disclosure Statement
All authors declare that they have no conflicts of interest.
Funding Information
This research was supported by National Natural Science Foundation of China under Grant Nos. 32172893 and 32302904, by Zhejiang Provincial Natural Science Foundation of China under Grant No. LQ22C180003, by Zhejiang Province Science and Technology Cooperation Project of “Three Rural and Nine Parties” under Grant No. 2024SNJF051, by “Pioneer” and “Leading Goose” R&D Program of Zhejiang under Grant Nos. 2023C02047, 2023C02023, and 2025C01138, by Science Foundation of Zhejiang Sci-Tech University (ZSTU) under Grant No. 20042220-Y.