Although adjuvants are commonly used in the formulation of billion-dose vaccines, the mechanisms of action are still poorly understood. Thus, a deep understanding of the way of action and the immunological mechanisms involved in the immune system response to pathogens represents a crucial step for the development of new adjuvants. Recently, significant attention has been paid to a deeper understanding of how vaccination adjuvants stimulate the immune response. Thanks to the recent advances in immunological research, it has been possible to elucidate some of the mechanisms by which adjuvants act, such as the depot effect and the release of cytokines and chemokines, the mobilization of immune cells at the injection site, the induction of adaptive immune responses, the increase in the antigen immunogenicity, and the activation of antigen-presenting cells (APCs) ^[14][15]. Clarifying all the mechanisms by which adjuvants explicate their action will furnish crucial information on how adaptive immunity is promoted by the innate one, and help in the development of new potent vaccines. Adjuvants can be classified using a broad range of factors, including their physicochemical characteristics, origins, and modes of action; one of the most popular classification schemes divides vaccine adjuvants into two main groups, delivery systems and immunostimulators. Another class of adjuvants is represented by the mucosal ones which can act both as delivery vehicles or immunostimulatory compounds, such as chitosan and its derivatives (N-trimethyl and mono-N-carboxymethyl chitosan), cholera toxin (CT), and the heat-labile enterotoxins (LTK3 and LTR72). Novel delivery system adjuvants are reported in Table 1. Traditionally, delivery vehicles operate only as a depot for immunostimulatory adjuvants to activate cells of the innate immune system cells. Since there is now evidence that some delivery mechanisms can activate innate immunity, this classification is no longer accurate ^[16].

In fact, delivery vehicle adjuvants both work as antigen carriers and cause a local pro-inflammatory response by activating the innate immune system, resulting in the recruitment of immune cells to the injection site. The antigen-adjuvant complex induces the activation of pattern recognition receptor (PRR) pathways by acting as pathogen-associated molecular patterns (PAMPs). These phenomena lead to the induction of innate immune cells, resulting in the release of cytokines and chemokines, the same mode of action exploited by immune potentiators adjuvants ^{[1][17][18][19]}. Immunoadjuvants (Table 1) are immune potentiator compounds that enhance antibody production by direct stimulation of the innate immune system. Moreover, adjuvants acting as immunomodulators can stimulate the production of specific types of cytokines, thereby boosting the response of the immune system. For example, alum, Freund’s adjuvant, and CpG oligodeoxynucleotides have been reported to induce the production and release of some cytokines involved in the regulation of innate and adaptive immunity, such as interferons (IFNs), interferon-γ (IFN-γ), and interleukins (IL2 and IL12) ^[2]. Several cytokines have been reported to act as immune potentiators adjuvants stimulating antigen-specific serum/mucosal antibody and cell-mediated immunity. Among this family of substances, the most well-known cytokines adjuvants are granulocyte/macrophage colony-stimulating factor (GM-CSF), IFN, chemokines, and a few interleukins (IL-1, IL-2, IL-12-IL-15, IL-18) ^[15]. Moreover, the immunostimulatory adjuvants are useful for the recruitment of immune cells, such as macrophages, neutrophils, and dendritic cells (DCs); the activation of the APCs; and the prolonged accumulation of the vaccine in the site of injection. Recent studies have linked Toll-like receptors (TLR) to autoimmune systems, discovering the mechanisms by which TLR activate the innate immunity system that results in adaptive immunity and inflammatory response induction, ensuring long-lasting protection ^[20].

Adjuvants can act as delivery systems, entrapping, adsorbing, or aggregating antigens and slowly releasing them over time. This mechanism, defined depot effect (Figure 1a), occurs at the injection site where adjuvants prevent the removal of the antigen due to hepatic clearance; this enhances the vaccine’s half-life and ensures a continuous stimulation of the immune system resulting in high antibody titers. Over the years, many examples of adjuvants acting through the depot effect have been described, such as liposomes, emulsions (both o/w and w/o), virosomes, and lipid or polymeric nanoparticles (NPs). Some of them have been developed to simulate pathogen membranes to transport, preserve, and release the antigens, and simultaneously enhance their immunogenic functions. Several types of liposomes, such as the traditional ones, the multilamellar vesicles (ICMVs), or the solid core liposomes, exploit their action also by promoting the depot effect.

Water in oil emulsions, such as the complete Freunds adjuvant (CFA), and some NPs also act through the depot effect that ensures long-lasting immune responses ^[2][16]. Particulate adjuvants can induce immune responses by exploiting several mechanisms, such as up-regulating the release of cytokines and chemokines, inducing an inflammatory state at the injection site that activate the inflammatory cascade and recruiting innate immune cells. For example, the oil in water (o/w) emulsions MF59 and AS03 stimulate the recruitment of immune cells (neutrophils, monocytes, macrophages, and DCs) that transport both the antigen and the adjuvant to closer lymph nodes. The recruitment of immune cells in the injection site induces the activation of caspases, resulting in a further release of chemokines (IL-18, IL-33, IL-1β) which attract other DCs and prolong this phenomenon (Figure 1b). Furthermore, MF59 and AS03 increased at the site of injection the expression of CCR2, leukocyte-recruiting chemokines (e.g., CCL2, CCL3, and CCL5), as well as colony-stimulating factor 3 (CSF3). Similarly, alum induces a local pro-inflammatory microenvironment after injection that provokes the activation of the complement cascade leading to the recruitment of immune cells from the bloodstream ^[2][16]. Inflammasomes represent an important component of the innate immune system. They are required for an effective immune response to pathogens. When an inflammasome is activated, the cell secretes pro-inflammatory cytokines, such as IL-18, IL-33, and IL-1β, which boost the adaptive immune response (Figure 1b). Inflammasomes are cytosolic protein signaling pathways made of working components, such as a leucine-rich repeat (LRR) C-terminal or DNA-binding domain (HIN200), a caspase-1 effector and an adaptor protein ASC which activate inflammatory caspases. Granulocytes, T- and B-cells, monocytes, hepatocytes, neurons, microglia, and Langerhans cells all express inflammasomes that are responsible for recognizing pathogens and initiating an innate immune response. When an inflammasome is activated, it proteolytically cleaves pro-caspase 1, liberating the active form which converts pro-IL-1β and pro-IL-18 into the active species. Released from cells, ILs initiate inflammation and induce the immune response that protects against pathogens. Furthermore, IL-18 activates lymphocytes and stimulates the proliferation of T-cells and B-cells, the activity of natural killers (NKs), and the secretion of IFN-γ, TNF, IL-1, and IL-2. Thus, adjuvants acting as inflammasome activators represent successful strategies to enhance and sustain immune response strength. These adjuvants activate inflammasomes through similar mechanisms, including degradation of lysosomes, cathepsin release, and the formation of reactive oxygen species (ROS). Among the inflammasome activators, adjuvants, such as aluminum salts, chitosan, saponins, flagellin, and synthetic cation polymers can be found. Aluminum salts provoke lysosome damages which induce the production of cathepsin B involved in the formation of the inflammasomes, in particular the NOD-like receptor protein 3 (NLRP3); the active inflammasome triggers caspase-1 and stimulates the release of cytokines. Chitosan and nanoparticles (NPs) made of synthetic cation polymers activate the NLRP3 inflammasome and enhance the secretion of several interleukins (IL-2, IL-4, IL-6, IL-10, IL-17A, and TNF), IFN, and IgG titers, boosting both cellular and the humoral immune responses ^[21][22]. Adjuvants can boost the immune reaction to vaccines through a wide range of mechanisms, such as depot effect and the stimulation of innate immunity. The first line of defense against pathogens is represented by the innate immunity. In fact, early recognition of pathogens is a key step in developing adaptive immune responses. Adjuvants can induce innate immunity by activating cellular pattern recognition receptors (PRRs), which recognize PAMPs and damage-associated molecular patterns (DAMPs) and stimulate APCs. Due to the central role in the innate immune system, PRRS represents a strategic target for new adjuvants. Within the PRRs superfamily, TLR, distinguished into surface and endosomal receptors, are promising adjuvant targets because they can induce signaling pathways, resulting in the induction of key transcription factors, such as nuclear factor-B (NF-B). Adjuvants can also be used to target endosomal PRRs, such as nucleotide-binding oligomerization domain-like receptors (NLRs) and retinoic acid-inducible gene-I-like receptors (RLRs) (Figure 2a). The localization is strictly related to their properties; in fact, plasmatic TLR recognize pathogenic proteins and lipids, while endosomal ones are activated by nucleic acids. TLR induce NF-kB through the MyD88 pathway, resulting in the release of pro-inflammatory cytokines. TLR-based adjuvants replicate PAMPs produced during the infection and can, thus, be extremely effective against pathogens or diseases that normally induce PRRs. Despite the excellent immunostimulatory efficacy of PRR agonists, their use as vaccine adjuvants has limitations due to high manufacturing costs which represent a limit for future clinical applications ^{[16][23][24][25]}. APCs, such as dendritic cells (DCs), express a variety of PRRs that allow them to recognize several pathogenic constituents. When PRRs are activated by PAMPs, they initiate complex signal cascades that result in the production of cytokines and chemokines, which include interferons (IFNs), the enhancement of antigen presentation capacity, and the migration of DCs to lymphoid tissues, where they interact with T cells and B lymphocytes to initiate and shape the adaptive immune response. Matured DCs can also stimulate naive CD4+ T cells to differentiate into different T helper (Th) subsets (e.g., Th1 and Th2 cells), which help B cells produce antibodies. Several cytokines regulate Th cell differentiation; for example, cytokines such as IL-12, IL-15, and IL-27 regulate the development of naive CD4+ T lymphocytes in Th1 cells. In summary, Th1 cells predominate in response to intracellular pathogens, such as viruses and some bacteria, whereas Th2 cells predominate in response to large extracellular parasites ^[23]. DCs are also able to stimulate naïve cytotoxic CD8+ T cells into activated CD8+ T cells ^[26]. This phenomenon called “cross presentation” is necessary for inducing strong and durable cellular immunity against exogenous antigens, and for the effective prevention of viral diseases and cancer. It is still unclear how exogenous antigens are processed in DCs and presented to CD8+ T lymphocytes on MHCI; however, two different mechanisms have been proposed ^[27]. In the cytosolic pathway, antigens enter into the cytosol through endosomal vesicles, and are degraded by proteasome. In the vacuolar pathway, antigens are degraded in lysosomal compartments, independently from proteasome activity. Aluminum, saponins, and TLR adjuvants can act using this mechanism.

Antigen presentation elicited by the major histocompatibility complex (MHC) on APCs, represents a critical step in the activation of adaptive immunity. Many adjuvants, such as alum, emulsions, and NPs, were supposed to function by “targeting” antigens to APCs, enhancing the antigen presentation by MHC. To date, it has not been clarified yet whether the mechanism through which adjuvants increase antigen presentation contributes to the development of the adaptive immune system. For instance, alum has been shown to boost the antigen uptake by DCs, as well as prolong the duration of antigen presentation. Antigen size appears to be important in modulating antigen presentation efficiency. Large lipid vesicles are found in early endosomes/phagosomes, where they increase antigen presentation, whereas smaller vesicles are found in late lysosomes, where they decrease antigen presentation ^[16].