Subunit vaccines contain selected immunogenic components of the pathogen to elicit an immune response (1, 2). Particulate vaccines involve the attachment of the antigens to microcarriers through chemical adsorption, encapsulation, conjugation, or biological self-assembly for enhanced delivery and induction of an efficient immune response (3, 4). Soluble vaccines are weakly immunogenic when compared to their insoluble counterparts such as when displayed on particulate carrier (5). Due to low immunogenicity, soluble vaccines often require the administration of multiple boosts (6, 7). The use of immunostimulatory adjuvants along with soluble vaccines would improve the immune responses but potentially increases the overall vaccine production cost (8). However, antigens immobilized on particulate carrier exhibit enhanced immunogenicity (9–11). Some particulate carriers themselves act as adjuvants resulting in enhanced and targeted immune responses. Particulate carrier also allow the codelivery of adjuvants and multiple antigens to the same antigen presenting cells (APCs) (12, 13). Therefore, particulate vaccines are promising and potent antigen delivery systems to overcome the low immunogenicity of soluble subunit vaccine formulations.

The proposed mechanism of antigen processing elicited by soluble and particulate vaccine formulations is illustrated in Figure 1 . Soluble antigens can be internalized via endocytosis (14) and exclusively presented by major histocompatibility complex (MHC) II machinery in the endosomes, whereas particulate antigens above 500 nm can be phagocytosed by APCs into the phagosomes and presented by both MHC class I and II machineries in the cytosol (7). The maturation of the phagosome occurs after the formation of nascent phagosomes (pH 7.4) containing the engulfed particulate antigens which are then sequentially trafficked into progressive acidified compartments called early phagosome (pH 6), late phagosome (pH 5.5), and phagolysosomes (pH 5) (15). The efficient degradation of antigens into smaller peptides occurs in the phagolysosomes which contain a variety of digestive enzymes such as proteases, lipases, and glycosidases without degrading the epitopes (16). Further degradation of antigens into smaller polypeptides occurs in the cytosol by a protein complex called proteosomes (17). The degraded protein fragments will be transported into the endoplasmic reticulum, where the folding and assembly of the heavy and light chain of MHC molecules occurs and facilitates the MHC and peptide binding (18). Finally, the peptide-loaded MHC complex gets transported to the cell surface through the Golgi apparatus and attracts both CD4⁺ T cells and CD8⁺ T cells with specific receptors to mount a cell-mediated immune response eliminating the damaged/infected cells displaying corresponding peptide fragments (19). MHC class I and II pathways are usually involved presenting peptides from intracellular or extracellular pathogens, respectively. However, dendritic cells (DCs) possess the ability to divert peptides derived from extracellular pathogens to cytotoxic CD8⁺ T cells via the MHC class I presentation pathway (20). A phagosome is a key organelle in antigen cross-presentation which primarily induces cytotoxic CD8⁺ T cell responses. However, such responses mediated by the phagosome route are not achievable by endocytosed soluble antigens (21). In addition, B cell receptors (BCRs) are uniformly distributed on B cell surface in the absence of pathogen invasion. However, BCRs are brought together to bind multiple copies of antigens on the invading pathogen’s surface. This process is also called BCR cross-linking, required for B cell activation. Repetitive display of antigens on particles facilitates efficient recognition and BCR cross-linking, which allow strong B cell activation and antigen uptake for presentation to CD4⁺ T cells. It results in inducing higher levels of neutralizing antibodies and functional cellular immune responses than achieved by soluble antigens present in most subunit vaccines (22).

Soluble antigens are engulfed by APCs through endocytosis and get presented to endosomes. Consequently, endosomes fuse with lysosomes and degrade the antigens into peptide fragments restricted to both MHC class I and II. MHC class II specific peptides bind to MHC class II molecules by replacing the class II-associated invariant chain peptide (23). This peptide-MHC class II complex will get presented to the cell surface and activates naïve CD4⁺ T cells. However, if the peptides are MHC class I specific, they will get cross-presented to proteosomes and follows MHC class-I pathway to expand cytotoxic CD8⁺ T cells (14). Even though the cross-presentation of soluble antigen was detected in vivo, it is not very efficient in generating strong immune responses (24). However, unlike soluble antigens, particulate antigens are sustainably presented by APCs in large quantities for a prolonged time resulting in enhanced immunogenicity (25).

The ultimate aim of vaccination is to induce protective immunity against pathogens (10). The addition of adjuvants can further enhance and/or modulate the immunogenicity of particulate vaccines resulting in enhanced vaccine performance (1, 84). Induction of protective immunity requires effective priming of T and B cells (10, 59, 87). The activation and effector phases of T cell-mediated responses require two signals, “signal one” through T cell receptor recognition of peptide-MHC complexes and “signal two” via costimulatory receptors on T cells. Both signals are provided by APCs that encounter pathogens (1). Adjuvants can stimulate the activation and maturation of APCs, such as DCs, and thus enhance the expression of MHC and co-stimulatory molecules, which are essential for the induction of adaptive immunity (1, 10). Adjuvants are usually categorized as the delivery system (such as lipid vesicles) or immunostimulatory molecules (such as PRR ligands) based on their proposed mechanism. Most adjuvants possess both properties (88). For example, alum salts promote Th2-type immunity and B cell differentiation, leading to tremendous antibody production (78). Adjuvant System 04 (AS04) is a combination adjuvant, consisting of alum salts and TLR4 ligand monophosphoryl lipid A, which respectively upregulate potent humoral (Th2) and cellular (Th1) immune responses ( Figure 2 ) (88).

As PRR ligands enable innate recognition by PRR, such as TLRs, present on DCs, the fabrication of particulate vaccines with PRR ligands can lead to targeted delivery to DCs and enhance the effectiveness of particulate vaccines (89). This enhancement is achieved by inducing “signal two” through the expression of co-stimulatory molecules on DCs, which augments T-cell responses (89). Thus coupling PRR ligands with antigens confer innate activation and antigenic stimulation to the same DC that uptakes the particulate vaccine. This lead to the generation of both signals, MHC-peptide complex and co-stimulatory signal, required for T-cell activation (1). In addition, targeted delivery to DCs can alternatively be achieved through the co-delivery of the CD40 ligand with antigen. The ligation of the CD40 ligand with the CD40 receptor on DCs serves as a key co-stimulator for DCs maturation and induction of CD4⁺ T cell responses (90).

The ability of DCs to present extracellular antigens in the context of MHC class I, a phenomenon called cross-presentation, can be an ideal target for the targeted delivery of vaccine antigens (20). Adjuvants such as MF59 and QS-21 in combination with TLR ligands can facilitate cytosolic or vacuole pathways of cross-presentation (91). In addition, cell-penetrating peptides (CPPs) also known as protein translocation domains or membrane translocating sequences, are small peptides with strong membrane permeability. CPPs are comprised of 6 to 30 amino acid residues and majority of them are basic amino acid residues, leading to an overall positive net charge (92–94). Recently the use of arginine- and lysine-rich CPPs in conjunction with particulate vaccines promotes the MHC class I pathway cross-presentation effectively against viral and tumor antigens (20, 92, 93). Moreover, DC targeting peptides (DCpep) are strongly targeted to DCs. They can improve vaccine capture efficiency, and promote DCs maturation, cytokine secretion, and T cell proliferation. Vaccines with DCpep can significantly induce stronger immune responses than the vaccines without DCpep (95). For example, Clec9A is produced on various DC subsets, such as mouse CD8a⁺ DCs and CD103⁺ DCs, and responsible for antigen cross-presentation. CBP-12 has high affinity with Clec9A on DCs. Vaccines with CBP-12 has been shown to elicit both strong cytotoxic CD8⁺ T cell and antibody responses (96, 97).

Post-translational modifications (PTMs) regulate function of proteins including antigens and contribute to their immunological properties and stability (98). A study showed that PTMs are often required for antigens to induce functional immune responses (98). Over the last decade, an increasing number of PTMs have been detected and characterized in E. coli, and most PTMs are rarely found in bacterial antigens with the majority of modified antigens having a low sub-stoichiometric degree of modification (99). As a result, retaining the conformation and function of eukaryotic multi-domain antigens produced in E. coli is more challenging (100).

Although the E. coli expression system is commonly used to produce recombinant protein vaccines, mammalian or insect cells should be considered for antigens that require high levels of PTMs that are required for vaccine purposes (98, 101). These post-translationally modified complex antigens can then be incorporated into particulate platforms using a variety of methods ( Figure 2 ). Non-covalent methods include the use of peptide tags like polyhistidine, protein tags such as maltose-binding protein and glutathione- S-transferase, DNA-directed immobilization, and the biotin–streptavidin pair (102). One of the most powerful non-covalent biological interactions known is the binding of biotin to avidin (103). Purified post-translationally modified complex antigens that have been biotin labelled are therefore highly effective for protein capture on particulate platforms for vaccine delivery, and the biotinylated antigens are subsequently recognized by avidin/streptavidin. Although chemical biotinylation frequently results in heterogeneous products with impaired function (104), enzymatic biotinylation with E. coli biotin ligase (BirA) attaches biotin to the 15 amino acid avidin tag (AviTag) peptide yielding a homogeneous product with a high yield (105). Streptavidin is used as a potent immunostimulant in less immunogenic antigen-based vaccines, most notably cancer vaccines, in addition to binding to the biotinylated antigen (106).

Although the interaction between streptavidin and biotin is strong, the binding can be destroyed by molecular motors (such as FtsK) in seconds (107) or shear forces in milliseconds (108), making it challenging to implement barriers or locks in cellular systems. Vaccine stability is required not just during storage but also following injection, where the lower concentration and other circumstances, such as endosomal pH and shear stress in the circulation, may mediate the dissociation of non-covalently attached antigens from particles (109). Therefore, covalent interaction between the target antigen and carriers is considered as a distinct, more robust, and long-lasting method of attaching antigens to the surface of an antigen carrier. Covalent linkage to peptide tags can be accomplished using SortaseA, SNAP-tag, split inteins, HaloTag, click reactions and Electrostatic Interaction Locks; however, SpyCatcher/SpyTag technology is most commonly used in vaccine delivery because SpyCatcher forms intermolecular isopeptide bonds selectively and spontaneously with SpyTag without the need for additional enzymes or chemical catalysis (102, 105). Furthermore, in comparison, SpyCatcher/SpyTag chemistry is reactive at the terminal and internal sites of a protein and can improve protein stability without changing its function (110). Thus, the bacterial production host can be bioengineered to produce vaccine particles displaying SpyCatcher to enable specific immobilization of SpyTag-fused target antigens with proper PTMs produced by such as mammalian or insect cell cultures (111, 112). However, several limitations typically may restrict the use of SpyCatcher/SpyTag in vaccine delivery. The final construct contains around 17 kDa molecular scar left by SpyCatcher/SpyTag coupling unlike the sortaseA with a smaller scar or split intein with no scar. Moreover, SpyCatcher/SpyTag is derived from Streptococcus pyogenes. The potential immunogenicity problem related to its bacterial origin may be an issue for vaccine design (113).

Antigens delivered in particulate form show superior immunological properties when compared to corresponding antigens in soluble forms. APCs, such as DCs, can cross-present antigens taken up in particulate form to potently activate both cytotoxic CD8⁺ and CD4⁺ T cells. Particulate vaccines are versatile as they can be formulated with adjuvants and/or bioengineered for the co-delivery of antigens with PRR ligands, CPPs, and DCpep for induction of protective immunity. Immobilizing antigens to particulate carriers significantly enhances their stability such as enabling the generation of ambient-temperature stable vaccine formulations. Bacterial production hosts are unable to produce complex antigens with high levels of PTMs. This review highlighted the advances of using various technologies via covalent and non-covalent attachments to incorporate antigens with PTMs on particles. Although particle vaccines possess a great promise to combat infectious diseases, there are still a number of unknowns. These include a profound understanding of how particle size, charge, and structure influence the induction of immune responses. Safety concerns, such as severe anaphylaxis and myocarditis, have also been raised due to the extensive use of some new nanoparticle vaccines, such as mRNA-LNP. Understanding the underlying mechanisms of nanoparticle vaccine properties and potential toxicity could significantly advance the rational design of prophylactic and therapeutic nanoparticle vaccines.

All authors contributed to the manuscript. SC and BR conceived and revised the manuscript. All authors contributed to the article and approved the submitted version.