Abstract
Over the past 20 years, a variety of potential adjuvants have been studied to enhance the effect of oral vaccines in the intestinal mucosal immune system; however, no licensed adjuvant for clinical application in oral vaccines is available. In this review, we systematically updated the research progress of oral vaccine adjuvants over the past 2 decades, including biogenic adjuvants, non-biogenic adjuvants, and their multi-type composite adjuvant materials, and introduced their immune mechanisms of adjuvanticity, aiming at providing theoretical basis for developing feasible and effective adjuvants for oral vaccines. Based on these insights, we briefly discussed the challenges in the development of oral vaccine adjuvants and prospects for their future development.
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Biogenic adjuvant materials used in the study of oral vaccines include bacteria-derived adjuvants, biologic proteins or peptides, intestinal immune cells targeting peptides, and some small-molecule immunomodulatory proteins. |
Non-biogenic adjuvant materials used in oral vaccine studies mainly involve biodegradable polymers {such as poly(d,l-lactide-co-glycolide) [PLG], poly( d,l-lactic-co-glycolic acid) [PLGA], chitosan and their derivatives, alpha-galactosylceramide [α-GalCer], ulex europaeus agglutinin-1 [UEA-1]}, and some synthetic toll-like receptor agonists and their derivatives. |
The combination of multitype materials has been used to design oral adjuvants; some protein vaccines (or biogenic adjuvants) are usually coated (or capsuled) with polymer-based microparticles/nanoparticles to prevent degradation in mucosa; some biogenic adjuvants are usually combined with the engineered living intestinal beneficial bacteria as a carrier to construct oral vaccine candidates. |
1 Introduction
Pathogens initiate infection mainly by accessing the mucosal surface of the host, especially from oral-to-gastrointestinal tract (GIT). It is generally considered that direct vaccination via the mucosal surface at the initial site of infection is the most effective way to trigger protective mucosal immune response against pathogens [1,2,3], but the vast majority of vaccines are administered by injection [2]. Compared with parenteral vaccination (or traditional injection), peroral vaccination or administration requires less stringent regulatory requirements, allowing for the self-administration of oral formulations. For humans, oral vaccination will minimize the need for trained healthcare personnel [4, 5] and eliminate occupational needle-stick injuries, which could reduce blood-borne infectious diseases such as acquired immune deficiency syndrome (AIDS)/human immunodeficiency virus (HIV) and hepatitis [6, 7]. For animal husbandry and aquaculture, the use of oral vaccination for disease prevention and control can reduce the labor cost of animal management and reduce the stress response of animals. Therefore, oral vaccination is potentially easier, safer, more convenient, more time-saving, and more economical [8].
However, oral vaccination is still challenging because most oral vaccinations universally could not trigger sufficient immune response, mainly because of an inadequate specific secretory immunoglobulin A (sIgA) response. There are two main reasons for this dilemma. First, the harsh conditions of the gastrointestinal environment, including hydrochloric acid, digestive enzymes, bile salts, mucus, antimicrobial peptides, and gastrointestinal peristalsis, would cause low bioavailability of antigens, which leads to acquired immune tolerance instead of stimulation [9,10,11,12]. Second, antigen-presenting cells (APCs) residing in the GIT are tolerogenic and hyporesponsive [13]; therefore, substantial impediments exist for oral vaccines to reach the inductive site of the mucosa-associated lymphoid tissues (MALT) and to trigger immune response, which critically hinders the effectiveness of oral mucosal immunization.
Given the poor immunogenicity (or low bioavailability) of oral vaccines, using appropriate and effective oral mucosal adjuvants may be critical to the success of peroral mucosal vaccination. Typical adjuvants, such as alum, complete Freund’s adjuvant (CFA), and incomplete Freund’s adjuvant (IFA), etc., have long been used in injectable vaccination but they do not work well in peroral mucosal immunity. To successfully stimulate intestinal mucosal immunity, oral vaccine adjuvants need to have two main properties—GIT delivery stability and intestinal mucosal adjuvanticity—because they must find an effective way to deliver vaccines (or antigens) to the dendritic cells (DCs), macrophages, and lymphocytes located in MALT through these natural barriers in GIT, and exert their adjuvant properties, while protecting the loaded vaccines (or antigens) from the harsh peroral mucosal environment. Therefore, it is necessary to explore effective peroral mucosal adjuvants to improve the effectiveness of oral vaccines; however, to date, no adjuvants have been included in licensed oral vaccines [14].
In recent years, many researchers have focused on finding safe and effective adjuvants (or delivery systems) to formulate oral vaccines, and have made great progress in the development of oral vaccine adjuvants. At present, some potential oral mucosal adjuvants have shown promising prospects, for instance modified bacterial enterotoxins (e.g., double-mutant heat-labile toxin [dmLT] of Escherichia coli and multiple mutant cholera toxin [mmCT]), some small molecule immunomodulatory proteins, and some non-biogenic biodegradable polymer materials {e.g., poly(d,l-lactide-co-glycolide) [PLG], poly(d,l-lactic-co-glycolic acid) [PLGA], alpha-galactosylceramide [α-GalCer], Ulex europaeus agglutinin-1 [UEA-1]}, and some synthetic toll-like receptor (TLR) agonists and their derivatives.
In this review, we systematically summarize various peroral adjuvant candidates, including the completed, ongoing, and planned study candidates. According to the physicochemical properties of these peroral adjuvant candidates, we classified the existing or emerging oral vaccine adjuvants as biogenic adjuvants (including bacteria-derived or biologic protein, peptide, or immunostimulants, small-molecule proteins, etc.), non-biogenic adjuvants (e.g., various biocompatible polymer-based microparticles/nanoparticles), and biogenic and non-biogenic composite adjuvants. We also introduced their general properties, mechanisms of adjuvanticity, origins and brief histories, preparation processes, and results of preclinical studies or even clinical studies, and discussed prospects for their application as oral vaccine adjuvants. This article reviews the research progress of oral adjuvants in recent years, aiming to promote the application prospects of oral vaccines.
2 Biogenic Type Oral Vaccine Adjuvants
At present, many effective oral vaccine adjuvants are still derived from biological material, such as bacteria-derived adjuvants (e.g., bacterial enterotoxins, bacterial flagellin, bacteria-derived enterocyte-targeting proteins, and some bacteria-derived proteins), protozoan-derived adjuvants, intestinal immune cells targeting peptide adjuvants, small molecular immunomodulatory proteins (SMIPs; e.g., cytokines and thymosin α-1 [Tα1]), Fc region of immunoglobulin (Ig) G, and adjuvants composed of multiple biogenic materials (Table 1).
2.1 Bacteria-Derived Adjuvants for Oral Vaccines
Targeting specific bacterial organelles or components, the host’s immune system has evolved to recognize infections and activate the most potent immune cells to fight the pathogenic bacteria. When developing vaccines, adding appropriate bacterial organelles or components into vaccines would produce a stronger immune response to provide better and more enduring immune protection against infections. Bacteria-derived adjuvants have attracted particular interest for the development of oral vaccines because specific bacterial organelles or components have a role as immune stimulators.
2.1.1 Bacterial Enterotoxin Adjuvants
The most well-studied mucosal adjuvants to date are still the adenosine diphosphate (ADP)-ribosylating bacterial enterotoxins, such as cholera toxin (CT) produced by Vibrio cholerae, heat-labile toxins (LT) produced by enterotoxigenic Escherichia coli (ETEC), as well as their mutants or subunits. Initially, CT and LT were not only highly effective mucosal adjuvants but they were also very toxic, which precluded their clinical application. However, much effort has been devoted to developing variants of these enterotoxins that are low or non-toxic but still retain their adjuvant activity [15], such as LT (R192G or single-mutant LT [mLT]) [16], dmLT [17], and mmCT [18]. These enterotoxins (CT and LT) and their mutants (or subunits) [dmLT and mLT] can increase the generation of antigen-specific IgA antibodies, T-cell responses, and long-lasting memory when coadministered with antigens through the mucosal or transcutaneous routes [16, 19]. CT, LT, and some LT mutants could increase antigen capture in the small intestine by promoting DC migration from the subepithelial dome (SED) to the follicle-associated epithelium (FAE) between 1.5 and 12 h after oral administration [20]. Additionally, preclinical research showed that LT, dmLT, CT, and mmCT can all significantly raise T helper (Th) 17 responses and thus increase antibody responses (Fig. 1a) [16, 18, 19, 21].
Concise mechanisms of oral adjuvants at intestinal mucosal sites. a Bacterial enterotoxin (dmLT, mmCT) targets GM1 receptors, promotes Th17 response, and subsequently induces antigen-specific IgA antibodies. b Bacterial flagellin increases TLR5 stimulation that activates the production of inflammatory cytokines and subsequently augments innate and adaptive immune responses. c1 M cell-targeting peptides (CKS9, Co1) specifically target and bind to M cells. c2 RANKL, increasing the number of M cells. c3 DC-targeting ligand (DCpep), specifically targets and binds to dendritic cells. d Small molecular immunomodulatory proteins (cytokines and Tα1) directly stimulate, attract immune cells, and induce immune response. e1 PLG and PLGA protect antigens from degradation in GIT, allow the sustained and extended release of encapsulated antigens, and enhance antigen uptake by APCs, and subsequently the delivery of these microparticle-containing APCs to specific lymphoid compartments. e2 CS and its derivatives (TMC, HACC) and e3 PAHs possess mucoadhesive properties and permeation-enhancing effects. e4 ALG possesses mucoadhesive properties. f UEA-1 specifically targets and binds to M cells. g α-GalCer activates the iNKT-cell. h CpG-ODN activates TLR9 on B-lymphocytes and DCs, stimulates antigen presentation and induction of antigen-specific immune response towards the Th1 phenotype. CS chitosan, PAHs polyanhydrides, ALG alginate, iNKT-cell invariant natural killer T cell
Double mutant heat-labile enterotoxin (dmLT) The most widely used and promising bacterial enterotoxin adjuvant to date is LT (R192G/L211A) or dmLT. In fact, dmLT is a genetically attenuated derivative of a wild-type ETEC heat-labile enterotoxin, which changes arginine to glycine at amino acid position 192 to disrupt the enzymatic and toxic activity of LT, and changes leucine to alanine at a potential pepsin-sensitive proteolytic cleavage site at amino acid position 211 [17]. This detoxified or attenuated form of LT retains its antigenicity and adjuvant properties. dmLT has been shown to be safe, well tolerated, and reasonably immunogenic in oral doses up to 100 μg in humans [22]. To date, dmLT has been an effective adjuvant that strongly potentiated the immune responses of various vaccines administered parenterally and mucosally against infectious pathogens (Table 1), e.g., Streptococcus pneumoniae [23], Helicobacter pylori [24, 25], tetanus toxoid [17], CT [18], and ETEC [26]. Noteworthy, when prophylactic immunization was performed with H. pylori lysate antigens, dmLT promoted strong B- and T-cell immune responses to H. pylori antigens and reduced the bacterial load in stomachs of H. pylori-infected mice [25]. Adding dmLT to an attenuated Salmonella-vectored ETEC vaccine improved its immunogenicity in mice [27]. Through preclinical studies, Holmgren et al. showed that adding dmLT to the multivalent ETEC vaccine (ETVAX) significantly improved both the anti-colonization factor (CF) and anti-LT responses following oral immunization [28]. Moreover, the phase I study of human volunteer trials proved that dmLT further enhanced the mucosal immune responses to CF antigens present in low amounts in this ETVAX vaccine [28, 29]. In addition, through clinical trials of human volunteers, Harro et al. demonstrated that the shedding of challenge strain (ETEC H10407) in those human volunteers orally administered ACE527 (the ETEC vaccine) and dmLT was tenfold lower than in those who received the vaccine alone, illustrating that dmLT can significantly contribute to vaccine efficacy to protect human volunteers against ETEC challenge [30]. In conclusion, dmLT is a well-tolerated and powerful mucosal adjuvant for coadministered antigens.
Multiple Mutant Cholera Toxin (mmCT) CT used to be an effective adjuvant, widely used to induce mucosal immune responses in animal models; however, the strong enterotoxicity of CT precludes its use in human or veterinary vaccines. The recently developed mmCT, which derived from CT with mutations in multi-sites in its A subunit and is fully resistant to proteolytic cleavage, is a strong, yet practically non-toxic novel mucosal adjuvant. Compared with native CT, the cAMP-inducing activity of mmCT decreased by >1000-fold [31]. Compared with dmLT, mmCT protein is more easily produced and purified in large quantities because mmCT is secreted from the extracellular medium of CT-deleted V. cholerae, while dmLT is located in inclusion bodies [19, 31]. mmCT possesses similar adjuvant activity and safety as dmLT, which promotes human Th17 responses via cAMP-dependent protein kinase A and caspase-1/inflammasome-dependent interleukin (IL)-1 signaling [18]. The study by Holmgren et al. [32] reported that intragastric immunization of H. pylori whole-cell vaccine (WCV) together with mmCT reduced the colonization of H. pylori in the stomach of mice by 50- to 125-fold, which was associated with rises in both the anti-H. pylori antibody responses of serum IgG and intestinal mucosal IgA and the responses of strong T cell and interferon (IFN)-γ and IL-17A cytokines. Moreover, its immune effect is similar to that of WCV together with CT, indicating mmCT, a non-toxic adjuvant, can replace CT as an adjuvant without loss in protective efficacy [32].
In conclusion, mmCT has no enterotoxicity but retains strong adjuvant activity, is economical and easy to be produced, and has great potential in designing oral vaccines.
2.1.2 Bacterial Flagellin
Flagellin, the main structural protein of bacterial flagella, is considered a pathogen-associated molecular pattern (PAMP). TLR5 can recognize flagellin, thus activating the production of inflammatory molecules, including chemokines and cytokines (Fig. 1b), and then triggering cellular immune responses, including DCs, through myeloid differentiation factor 88 (MyD88) signaling [33, 34]. In addition to TLR5 activation, flagellin can bind to cytosolic nucleotide binding oligomerization domain-like receptors, NLRC4, which activate the caspase-1 inflammasome [35]. TLR5 is extensively expressed in the lung, intestinal epithelial cells, monocytes/macrophages, and DCs [36], Because flagellin is easy to express, is stable and potently activates the adaptive immune response by binding to TLR5 [37, 38], it has attracted a lot of attention as a vaccine adjuvant. Oral administration of flagellin-based vaccines could induce effective immune protection in mice. In the study by Ren et al. [39], the H5N1 chimeric virus-like particles (VLPs) containing membrane-anchored FliC (FliC-VLP) were administered orally to mice, and the virus-specific IgG titers of immunized mice were tenfold higher than those of mice immunized with H5N1-VLPs lacking FliC, which significantly improved the protective immune response to lethal challenge from both homologous and heterologous H5N1 viruses. According to Zhou et al. [34], mice orally inoculated with LBNSE-Flagellin (the recombinant rabies viruses [rRABV] expressing flagellin of Salmonella enterica subsp.) could recruit/activate more DCs and B cells in the periphery, and trigger a stronger adaptive immune response (i.e., virus-neutralizing antibody level). LBNSE-Flagellin could shield more mice from LD50 challenge infection with rabies viruses strain CVS-24 compared with the parent virus LBNSE group. An innovative study by Girard et al. [40] revealed that plant-produced flagellin (flagellin of Salmonella typhimurium [FljB]) was a more potent and effective adjuvant for oral immunization. Beyond that, using plant-produced flagellin as an adjuvant for oral vaccine did not elicit an immune response against FljB.
By incorporating membrane-anchored flagellin into bacterial ghosts (BGs), it may be possible to create a more effective oral BG-based vaccine [41]. Moreover, synthesizing varied flagellins in an oral live bacterial vaccine strain is an attractive method for generating protective immunity. In the study by Eom et al., mice were shown to be protected against the virulent Salmonella SL1344 strain [42] after receiving an oral immunization with attenuated S. typhimurium BRD509 vaccine strain that expressed FliC and FljB flagellins (diphasic S. typhimurium has two flagellin genes—the flagellin in phase I is FliC and the flagellin in phase II is FljB [43]). In addition, it is another promising way to develop oral probiotic live vaccine strains or oral attenuated (or non-virulent) salmonella live vaccine strains by integrating heterogeneous antigens into the hypervariable region of flagellin of probiotic strain [44] or attenuated (or non-virulent) salmonella strain [45,46,47].
In summary, it is commonly acknowledged that flagellin can boost an antigen-specific immune response when used as an adjuvant. This will facilitate the development of flagellin-based vaccines that are safer and more effective, as well as their entry into oral clinical trials.
2.1.3 Bacteria-Derived Enterocyte Targeting Proteins
Expression of enterocyte binding proteins derived from some pathogenic bacteria on the surface of probiotic strains as adjuvants to deliver eukaryotic expression plasmids into host intestinal epithelial cells could be an effective oral DNA vaccine strategy [36]. Internalin A (InIA) in Listeria monocytogenes (L. monocytogenes) and fibronectin binding protein A (FnBPA) in Staphylococcus aureus (S. aureus) are well-known enterocyte targeting proteins. InlA is a cell wall protein that allows L. monocytogenes to bind to and be internalized by epithelial cells [48]. And FnBPA is an epithelial cell binding protein that can bind to fibrinogen, elastin, and fibronectin allowing for internalization of S. aureus into non-phagocytic cells [49]. When InlA (or mInlA, the mutated form of InlA [Ser192Asn and Tyr369Ser]) [50] and/or FnBPA [51,52,53] were expressed on the surface of lactic acid bacteria (LAB) strains, these recombinant strains acquire the ability to invade mammalian cells through the interaction between InlA and/or FnBPA and cellular receptors, resulting in the increase of targeted antigens cDNA in the intestinal lumen and the enhancement of host immune response [50,51,52,53].
2.1.4 Other Bacteria-Derived Proteins
Rarely investigated as mucosal adjuvants, some bacterial proteins and messengers still lack a clear understanding of how exactly they trigger immunity. However, they could be candidates for oral vaccine adjuvants because of their capacity to facilitate the immune response to antigens.
Muramyl dipeptide (MDP) is part of the bacterial cell wall and is delivered as a dipeptide with tuftsin, a biologically active compound [54, 55]. Although their roles in oral immune adjustment have not been fully elucidated, it has been demonstrated that MDP and tuftsin can activate APCs [55]. In the study by Jiang et al. [54], the fusion protein of MDP and tuftsin was utilized as an adjuvant to modify the Lactobacillus casei vaccine strain. The results showed that antibody and T-cell responses were improved after oral administration in BALB/c mice.
PorA is an outer membrane protein (OMP) from the Neisseria meningitidis [56]. It is remarkable that PorA has an important feature of oral protein adjuvants, namely resistance to proteolytic enzymes in the GIT [57]. It has the potential to act as an oral adjuvant when conjugated to antigens. For example, when PorA was fused with the H. pylori HpaA antigen and expressed in Lactococcus lactis, PorA could significantly enhance the antibody response against the HpaA antigen after oral administration in mice [57].
3′5′-Cyclic di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger that has strong mucosal adjuvant activity and numerous effects on the immune system, including type I IFN responses, promotion of Th1 and Th2 responses, increasing lymphocyte proliferation, and activation of APCs [58, 59]. Oral administration of recombinant L. lactis strains co-producing c-di-AMP and an anti-Trypanosoma cruzi antigen resulted in a T. cruzi-specific immune response
The Salmonella resistance to complement killing (RCK) protein plays an important role in interfering with complement killing and invading cells, including epithelial cells and APCs [60, 61]. The use of RCK as an oral adjuvant for the L. lactis vaccine strain successfully increased immune responses, conferring full protection against very-virulent infectious bursal disease virus (IBDV) challenge [62].
2.2 Protozoan-Derived Adjuvant Variant-Specific Surface Proteins
A novel oral adjuvant candidate could be achieved from parasitic protozoa, Giardia lamblia, which colonizes in the lumen of the upper small intestine of many vertebrate hosts. Serradell et al. [63] reported the variant-specific surface proteins (VSPs) from the Giardia lamblia surface can not only resist proteolytic digestion and extreme pH in GIT, as well as temperatures, but also stimulate host innate immune responses in a TLR-4-dependent manner. They constructed chimeric VSP-pseudotyped VLPs expressing hemagglutinin (HA) and neuraminidase (NA) of the influenza virus. These VSP-pseudotyped VLPs, but not plain VLPs, produced robust immune responses, protecting mice from influenza infection and HA-expressing tumors after oral immunization. This versatile oral vaccine adjuvant based on VSPs can be applied to antigens from different infectious agents or tumors and facilitate their use in remote areas where cold-chain for vaccine is not guaranteed.
2.3 Intestinal Immune Cells Targeting Peptide Adjuvants
Microfold cells (M cells), a unique subset of epithelial cells found in the epithelia covering MALT, such as Peyer’s patches, are used by the mucosal immune system to sample antigens in the GIT [64]. A variety of substances, including bacteria, viruses, and antigens, can be transported by M cells from the lumen to the underlying lymphoid tissues thanks to their great transcytotic ability [65,66,67,68]. Additionally, various antigens delivered by M cells can be sampled and captured by DCs positioned inside or beneath the epithelium [69, 70]. In addition, DCs can extend their probing dendrites into the lumen to sample commensal or microbial immunogens after passing through tight junctions to reach the gut epithelia [71]. These DCs subsequently migrate into the lymphoid follicles, where processed antigens are presented to B and T cells to sequentially trigger humoral (IgA) and T-cell immune responses [68, 72]. The aforementioned immunologic mechanisms of M cells and DCs can be exploited for the development of oral vaccine adjuvants. Therefore, targeting intestinal immune cells (such as M cells and/or DCs) is a promising strategy for developing oral vaccine adjuvants.
2.3.1 M Cell-Mediated Oral Adjuvants
In peroral mucosal vaccination, targeting M cells is considered a frontline prerequisite for effectively inducing antigen-specific immunostimulatory effects [73]. In the GIT, M cells are the antigen-collecting portals located on the FAE of Peyer’s patches and the gut-associated lymphoid tissue (GALT) of different species, which facilitate to transport antigens from gut lumen to the submucosal immune system [68, 74, 75]. M cells are believed to play a role in controlling gastrointestinal infection and immunity [73]. Therefore, M cell targeting might be a promising strategy for developing effective oral vaccine adjuvants [76].
M cell-targeting peptides Through phage display technology, Cho and colleagues [77] identified an M cell-homing peptide, CKS9 (CKSTHPLSC), which can facilitate the transcytosis of target antigen in M cells. In addition, according to Kim et al. [78], fusion of enhanced green fluorescence protein (EGFP) with another M cell-homing peptide, Col, could direct EGFP to bind to M cells and effectively transport it to mucosal immune induction sites to improve immune induction. Soon afterwards, with M cell-targeting peptides (Co1 or CKS9) as an oral vaccine adjuvant and LAB strain as an oral delivery vector, researchers tried to develop probiotic-derived oral vaccines against porcine diarrheal diseases, including porcine epidemic diarrhea (PED) [79] and swine dysentery [80], and obtained encouraging experimental outcomes after oral administration in mice.
In addition, the efficient uptake of antigens by M cells requires specific surface receptor molecules. Targeting the inherent receptors specifically expressed on the surface of M cells is another way to target M cells to deliver antigens to improve vaccine efficacy. Glycoprotein-2 (GP-2) is a glycosylphosphatidyl inositol anchoring protein that is specifically expressed on M cells and serves as a transcytotic receptor for luminal antigens [81]. Therefore, targeting GP-2 with specific ligands should increase antigen delivery to the immune initiation sites. Khan et al. [82] selected a GP2-binding peptide ligand, Gb-1, through phage library screening, which showed high binding affinity to GP-2. When fused with EGFP, Gb-1 significantly enhanced the uptake of EGFP by M cells compared with EGFP alone. Likewise, the Gb1-EGFP fusion induced effective mucosal and systemic immune responses after oral administration in mice. Therefore, exploiting the GP2-binding peptide Gb-1 for oral vaccine delivery would be a realistic approach.
Cytokine receptor activator of nuclear factor kappa B (NF-kB) ligand (RANKL). The proportion of M cells in intestinal epithelial cells is very low, accounting for approximately 1% of the total intestinal surface [68]. Therefore, if the number of M cells could be increased, it would be a promising technique to improve the effect of oral vaccines. It has been well-documented that the cytokine receptor activator of the nuclear factor Kappa B (NF-kB) ligand (RANKL) is a prevalent control factor for inducing M cells to differentiate from intestinal epithelial precursor cells by interacting with the cytokine receptor activator of NF-kB (RANK) expressed on the sub-epithelium of Peyer’s patches in the intestinal tract [83,84,85]. It has been proven that systemic administration of exogenous soluble RANKL (sRANKL) can correct the M-cell deficiency and uptake impairment in the Peyer's Patch [73]. In this regard, oral immunization by administering RANKL to induce the supraphysiological amount of M cells and then administering M cell-targeting antigens may be a viable approach to enhance the effect of oral vaccination.
A recombinant L. lactis IL-1403 producing and secreting soluble RANKL (sRANKL-LAB) constructed by Kim et al. could increase the expression of M cells in mice to be 1.51-fold higher than that in the untreated group through oral administration [83]. Maharjan et al. firstly administered intraperitoneally (or systemically) transmembrane RANKL (mRANKL) to mice and then delivered microparticulate antigen orally, which significantly increased the expression of M cells in FAE, showing similar effect as sRANKL-LAB [85]. They also demonstrated that RANKL-mediated transcytosis of antigens through M cells can enhance mucosal and humoral immunity. Choe et al. constructed RANKL-secreting L. lactis (LL RANKL) as an oral adjuvant for the aP2 subunit (soluble recombinant partial spike S1 protein from PEDV) vaccine loaded in hydroxypropyl methylcellulose phthalate (HPMCP) microspheres (HPMCP [aP2] plus LL RANKL) [86]. Their results showed that titers of virus-specific IgA antibodies in colostrum, and neutralizing antibodies in serum of sows vaccinated with HPMCP (aP2) plus LL RANKL increased significantly, and the survival rate of newborn suckling piglets delivered by sows vaccinated with HPMCP (aP2) plus LL RANKL was similar to that of piglets delivered by sows vaccinated with a commercial PED killed vaccine. These preclinical studies show that oral administration of RANKL is a promising adjuvant strategy, which could be used for effective oral vaccination and even oral therapeutic administration.
2.3.2 Dendritic Cell-Targeting Ligands
DCs represent the interface of the innate and adaptive immunity, and DCs play a pivotal role in priming T-cell immune responses against the inoculated antigen. Therefore, DCs are the major determinants of vaccination, so targeting oral vaccines to DCs is another strategy to enhance vaccination efficacy [87,88,89]. With DC-targeting peptides (DCpep, FYPSYHSTPQRP) as adjuvant, many researchers tried to utilize various LAB strains (including Lactobacillus plantarum, L. casei, Lactobacillus acidophilus, Lactobacillus saerimneri, L. lactis, etc.) as oral delivery vectors to develop oral vaccines for zoonotic or veterinary infectious diseases, such as Bacillus anthracis, and obtained good preclinical research results in animal model experiments of various diseases (Table 1). These research cases showed that modifying and specifically targeting a certain antigen to DCs can enhance antigen uptake.
2.4 Small Molecular Immunomodulatory Proteins
SMIPs are synthesized and secreted by a variety of tissue cells (mainly immune cells). They have many biological functions, such as regulating innate immunity and adaptive immunity, hematogenesis, cell growth, pluripotent stem cells and damaged tissue repair. To date, SMIPs used in the research of peroral vaccine adjuvants are mainly cytokines and Tα1.
2.4.1 Cytokine-Derived Oral Adjuvants
Cytokines are small proteins released by various cell types. Their functions are to stimulate, attract, and regulate the activity of immune cells (especially T cells), enhance the signal transduction of APCs, and sequentially improve the immune response to pathogens. They play a critical role in the regulation of innate and adaptive immunity [93, 94]. Cytokines have already been used orally to steer the immune system towards an increase in local cytotoxic T lymphocyte (CTL) activity and/or increased IgG and IgA titers. Some cytokines have been investigated as adjuvants for oral vaccines, and success has been reported in various preclinical studies, in which IL-2 is the most widely used oral adjuvant (Table 1). In particular, by genetically modifying probiotic strains (L. casei strain or Bacillus subtilis spores) to express corresponding host cytokines (such as IL-1β [95, 96], IL-2 [97,98,99], IL-6 [75], IL-12 [100], and granulocyte-macrophage colony-stimulating factor [GM-CSF] [101]), and oral coadministration with antigens or vaccine strains could significantly stimulate the production of specific antibody response in animals compared with the control groups. In some animal challenge tests, obvious protective immunity could be produced to fight against various infectious diseases, such as H. pylori infection [98, 99], Leishmania major infection [100], rabbit hemorrhagic disease (RHD) [97], and canine corona virus (CCV) [101]. Despite the above promising results, the potential safety concerns of cytokines need to be considered before using them as adjuvants [93]. According to the immunological properties of target antigens (or diseases), selecting specific and suitable cytokines as oral adjuvants needs to be based on the expected immune response of vaccination and its known influence on immune cells, but this is still one of the challenges of current immunological research. Overall, the optimal regimen of cytokines should be determined before starting clinical studies.
2.4.2 Thymosin α-1
Tα1 is a non-toxic immunomodified peptide hormone secreted by the thymus. It plays a very important role in cellular immune response by triggering T-cell maturation, augmenting T-cell function, developing antibody production, promoting reconstitution of immune defects, and increasing cytotoxic cells, Th1 and Th2 cytokine production, and IgG and intestinal sIgA production [102,103,104]. On account of its adjuvant attributes, by conjoining with the CSFV-E2 antigen and displaying it on the surface of L. plantarum, Tα1 could be used as an adjuvant of oral vaccine against classical swine fever virus (CSFV), which showed that Tα1 molecule adjuvant could enhance immune response and augment specific lymphocyte functions [102]. Therefore, Tα1 will be a promising adjuvant strategy in the development of an oral LAB vaccine [36].
2.5 Fc Region of Immunoglobulin G
As a potential adjuvant, the Fc region of IgG has attracted considerable attention. More and more evidence has demonstrated that fusion of the Ig Fc domain with the desired protein can facilitate dimerization of the protein, thus potently elevating the pharmacological and immunological characteristics of the protein [105,106,107], because the Fc region of IgG specifically binds to the FcRn (neonatal Fc receptor for IgG), which mediates IgG transport across the polarized epithelial cell lining on the mucosal surfaces [108]. As we know, IgG plays a predominant role in providing immune defense against foreign pathogens. Therefore, researchers have tried to target pathogenic antigens to FcRn as a new strategy to overcome intestinal epithelial barriers for mucosal vaccine delivery and drug therapy. Fc fusion proteins, or the recombinant proteins constructed by fusing the desired pathogenic antigens with the Ig Fc domain, have recently been utilized to produce vaccine candidates against infectious agents, including herpes simplex virus (HSV; gD-Fc) [109], pseudorabies virus (PRV) (gB-IgG2aFc) [110], HIV (Gag-Fc) [111], influenza A (H1N1) virus (3M2e-Fc) [112] and classical swine fever virus (CSFV) (E2-Fc) [113]. The aforementioned Fc fusion proteins could improve humoral and cellular immune responses by oral or intranasal immunization.
2.6 Biogenic Composite Oral Adjuvants
By combining two or more biogenic adjuvant materials to form a new composite adjuvant regimen, it is possible to improve mucosal immunity of target antigens in the intestine lumen. In this way, the advantages of each adjuvant could be fully utilized to enhance the overall immune effect. Until now, only the combination of intestinal immune cells targeting peptides and cytokines or the combination of two intestinal immune cells targeting peptides have been used as composite biogenic adjuvants in the development of oral vaccines, achieving good preclinical results. In particular, Li et al. [75] reported a novel biogenic composite mucosal adjuvant, IL-6-CKS9, which was a recombinant cytokine produced by conjugating an M cell-targeting peptide (CKS9) with the c-terminus of murine IL-6. Oral administration of recombinant L. lactis IL-1403 vaccine strain containing the above composite adjuvant promoted mucosal immune response. In addition, through combining the M cell-targeting peptide (Col) and DC-targeting peptide (DCpep) as a composite adjuvant, Ma et al. [114] genetically engineered a Lactobacillus vaccine strain that could target intestinal M cells and DCs and express COE antigen of PEDV. The recombinant strain efficiently induced anti-PEDV mucosal, humoral, and cellular immune responses in mice after oral administration. This suggests that the combination of Col and DCpep is a promising adjuvant strategy for oral probiotic vaccines. It is believed that more biogenic composite oral adjuvants will appear in the future.
3 Non-biogenic Oral Vaccine Adjuvants
Non-biogenic oral vaccine adjuvant materials are mostly polymeric microparticles/nanoparticles. They have many advantages, such as good biocompatibility, biodegradability, easy processing and modification, controllable surface properties, etc., and they could deliver and protect DNA and antigen protein of oral vaccines (or drugs) and control their release. Beyond that, they also possess mucosal absorptivity and immunostimulatory activity to activate or enhance immunity. Therefore, the application of non-biogenic adjuvant materials in oral vaccine research, and even in biomedical research, has become increasingly popular, showing great application prospects (Table 2).
3.1 Alum
Alum, also referred to as ‘aluminium salts’, encompass aluminium potassium sulphate, aluminium hydroxide, aluminium phosphate, and amorphous aluminium hydroxyphosphate sulfate [115]. Alum is one of the most widely accepted vaccine adjuvants and is a component of several licensed parenteral vaccines [116]. Kapusta et al. [117] reported oral administration with nanogram doses of alum-adjuvanted hepatitis B surface antigen (HBsAg) in mice-induced humoral immune response at the protective level. However, alum is unable to enhance cell-mediated Th1 or CTL responses, which are vital to control most intracellular pathogens [118]. Furthermore, alum is considered a poor inducer of mucosal immunity [37].
3.2 Polymer-Based Microparticle/Nanoparticle Oral Adjuvants
To overcome the harsh environment of the GIT, different types of polymer-based nanoparticles (including synthetic and natural polymers) have been widely studied for the preparation of various microparticle/nanoparticle vaccines (or nanoparticle adjuvants) for the GIT due to their biocompatibility, biodegradability, non-toxic nature, and ease of modification into desired shapes and sizes, as well as protecting the vaccine bioactivity from adverse situations [2, 119]. Polymer-based nanoparticle adjuvants are made of polymers such polyanhydride, poly (ethylene-glycol), PLG, PLGA, poly(lactic acid) [PLA], chitosan, alginate, and their derivatives, among others, and they have demonstrated enhancement of intestinal immune responses in vaccines for preventing various infections and treating various inflammatory diseases [7]. In this part, the application and research progress of polymer-based microparticles/nanoparticles as adjuvants for the peroral vaccines were reviewed.
3.2.1 Polyanhydride-Based Oral Adjuvant Materials
Polyanhydrides (PAHs), a class of synthetic biodegradable, non-cytotoxic, biocompatible polymers, are polymerized by methyl vinyl ether and maleic anhydride [120, 121]. PAHs are inherently highly reactive to water, thus leading to relatively rapid hydrolytic degradation, breaking down into carboxylic acids without cytotoxicity [121]. PAHs have been used in vaccine delivery systems for a long time, and polyanhydride nanoparticles (PNPs) are licensed for oral drug delivery in the UK [121,122,123]. In fact, PAHs are also a promising oral vaccine encapsulating material with the function of adjuvant and carrier. First, polyanhydride particles are cleaved in the gut to expose carboxylic acid groups that form hydrogen bonds with the hydroxyl groups of glycoproteins in the gut mucus, giving polyanhydride particles their mucoadhesive properties [124, 125]. Second, it has been reported that polyanhydride particles possess intrinsic adjuvant properties, which can activate APCs and regulate the immune responses [121, 126]. Furthermore, polyanhydride particles have been demonstrated to be able to provide sustained release of protein antigens via surface erosion [121, 125]. In addition, polyanhydride materials can be made into nano-encapsulated formulations by nanotechnology, which can exert better adjuvant effects. PNP-based vaccines have been shown to successfully encapsulate and release antigens, activate B and T cells, and induce both antibody- and cell-mediated immunity towards a variety of immunogens [127]. Moreover, PNPs act as agonists of various TLRs (TLR2, 4, and 5) [10, 126], innate immunity, complement system, and APCs to modulate the immune responses and induce long-lasting immunity [121, 126, 128]. Renu et al. reported that mucoadhesive PNPs could protect the vaccine cargo and deliver it to intestinal immune sites to elicit robust mucosal immunity and mitigate Salmonella colonization and shedding [125]. Overall, PNPs have potent immune adjuvant properties when administered orally and can target immune cells of chickens [125], mice [129, 130], rats [131, 132], and other animals.
3.2.2 Poly(d,l-Lactide-co-Glycolide) and Poly(d,l-Lactic-co-Glycolic Acid)
PLG is a biodegradable and biocompatible polymer [133]. Microparticles prepared from PLG have been proven to be effective adjuvants for a variety of antigens because microencapsulation of PLG can protect antigens from adverse degradation, allow sustained and prolonged release of antigens for a long time, and enhance uptake of antigen by APCs [134]. These APCs containing PLG-microparticles are then delivered to specific lymphoid compartments, such as the spleen and mesenteric lymph nodes, where they effectively present antigenic epitopes to T lymphocytes, especially Th1 and Tc, thus inducing strong specific cell-mediated immunity (Fig. 1e1) [134, 135], which is urgently needed for eliminating intracellular pathogens in host cells. Kim et al. reported that using H. pylori lysates encapsulated in PLG nanoparticles as an oral vaccine candidate could induce the H. pylori-specific mucosal and systemic responses in mice, and enhanced Th2-type responses [136]. Kofler et al. reported that the pulmonary and serum immune responses of BALB/c mice were enhanced by oral immunization with LW50020 encapsulated with PLG microspheres [137]. Ramya et al. used PLG microspheres as an oral delivery system for β-propiolactone inactivated concentrated rabies virus (CRV) and found that Th1-mediated cellular immunity was activated after oral administration of PLG+CRV in mice [138]. In addition, PLG microspheres also have many potential advantages in gene therapy [133].
PLGA nanoparticles are US FDA-approved biocompatible and biodegradable polymers, which are widely used in preclinical vaccine delivery. PLGA has the functions of delivery device, protection, sustained release of encapsulated antigen, and enhancement of antigen uptake during vaccination [139,140,141]. In addition, PLGA combined with pH-responsive materials can adapt to the extreme GIT more efficiently and has the potential to become an oral vaccine adjuvant. Tan et al. designed an acid-resistant PLGA nanoparticle (HP55/PLGA-CCF) using pH-responsive material, HP-55, which was an effective immunomodulator and an oral carrier to enhance the efficacy of subunit vaccines. Mice immunized with HP55/PLGA-CCF nanoparticles could induce high levels of urease-specific antibodies and memory T-cell responses [142]. As pointed out by Munang’andu and Evensen [143], adjuvants that serve as antigen delivery vehicles and immunostimulants are able to enhance antigen uptake by APC. Furthermore, PLGA has the above two inherent adjuvant properties [144]. PLGA NP-rOmpW (i.e., the outer membrane protein W [OmpW] of Aeromonas hydrophila encapsulated in PLGA nanoparticles) provided dose-dependent protection against A. hydrophila infection in Rohu (Labeo rohita Hamilton) after oral administration [140]. In general, the design of PLGA nanoparticles as an oral immune adjuvant is a promising strategy to improve antigen uptake and vaccine efficiency.
3.2.3 Chitin, Chitosan and Their Derivatives
Chitin particles possess TLR-2-dependent adjuvant activity and can augment the Th1, Th2, and Th17 antigen-specific immune responses when admixed with protein antigens [145]. Chitosan (CS), a deacetylated form of chitin, is a polysaccharide composed of N-acetyl-d-glucosamine and d-glucosamine [146]. Because of its low toxicity, excellent biocompatibility, biodegradability, antimicrobial activity, mucoadhesive properties, and permeation-enhancing effects, chitosan has been widely used as a potential excipient for the oral delivery of DNA, peptides, and live attenuated virus [147,148,149,150,151]; however, its limited mucoadhesive strength and low water solubility at neutral and basic pHs are considered as two major drawbacks of its biomedical applications. The chemical modification of chitosan results in quaternized chitosan [152] or its derivatives, such as N-trimethyl chitosan (TMC) [153], O-2′-hydroxypropyltrimethyl ammonium chloride chitosan (O-2′-HACC) [154], and mannosylated chitosan (MCS) nanoparticles [155]. This enhanced the mucoadhesive properties of chitosan. In addition, many researchers are trying to optimize chitosan nanoparticles and combine them with other nano-materials for composite adjuvants, which can promote a more efficient immune function and serve as a promising carrier for oral protein vaccine delivery [146].
3.2.4 Alginate and Its Derivatives
Alginate is a non-toxic, biodegradable, low cost, readily available polysaccharide copolymer containing (1-4)-linked β-d-mannuronate and α-l-guluronate residues, and is a mucoadhesive, biocompatible, non-immunogenic substance [2]. Alginate has been widely used in drug delivery because of its ability to contract in the stomach and release its cargo in the intestine. Alginate polymer as a single component is rarely used as an adjuvant. Alginate would usually be anchored/coated with chitosan or other electropositive materials by chemical modification to develop alginate-based composite adjuvant formulations for oral protein antigens (or vaccines) delivery (Tables 2, 3), such as alginate-coated chitosan microparticles (ACMs) [156] and alginate-chitosan coated layered double hydroxide nanoparticles (LDHs) nanocomposites (ALG-CHT-LDH) [157].
3.3 M Cell-Targeting Polymeric Particles (Ulex Europaeus Agglutinin-1)
UEA-1 is a lectin with specific binding activity to epitopes containing α-L-fucose [158]. UEA-1 can exclusively bind to M cells of mouse small intestine [159] and has been identified as an M cell-selective molecular marker [160]. Bioactive UEA-1 has been explored in the present investigation for targeted oral immunization. Gupta and Vyas reported UEA-1 conjugated liposomes as an oral M cell-targeted vaccine delivery vector [161]. In their study, the UEA-1 conjugated liposomes were predominantly targeted to the M cells. The serum anti-HBsAg IgG titer was obtained after oral immunization with HBsAg-encapsulated liposomes conjugated with UEA-1 for 3 consecutive days. The boosting immune effect was comparable with the titer recorded after single intramuscular immunization with alum-HBsAg [161]. Moreover, UEA-1-conjugated liposomes induced higher sIgA levels in mucosal secretions and cytokine levels in the spleen homogenates [161].
3.4 Alpha-Galactosylceramide
α-GalCer, a synthetic glycolipid, is a potent inducer of the invariant natural killer T (iNKT) cells, which are an important innate immune cell type [162]. α-GalCer can be presented by the CD1d molecules on the APC to NKT cells [163], which leads to activation and expansion of NKT cells, and subsequently induces full maturation of DCs in the spleen after immunization [162]. Therefore, α-GalCer is identified as a non-toxic oral adjuvant. It has recently been shown that α-GalCer acted as an oral active adjuvant to induce T-cell immunity against pathogenic bacteria and viruses through efficient activation/maturation of DCs. According to studies, α-GalCer potentiated mucosal immune responses to the HIV model envelope peptide (R15K peptide) [162], ETEC vaccine [164], V. cholerae vaccine [14], and whole-cell killed (WCK) H. pylori candidate vaccine [165] through oral immunization. The study by Davitt et al. demonstrated that α-GalCer was as effective as the ‘gold standard’ mucosal adjuvant CT in promoting intestinal IgA responses against a novel ETEC antigen [164]. In another study by Davitt et al., the addition of α-GalCer enhanced mucosal immunogenicity of Dukoral®, the most widely licensed oral cholera vaccine (OCV) internationally, and significantly increased intestinal anti-LPS and anti-cholera toxin B subunit (CTB) IgA responses against V. cholerae infections [14]. Longet et al. demonstrated that oral immunization of H. pylori WC antigen adjuvanted with α-GalCer significantly reduced bacterial loads in the stomach of H. pylori-infected mice; this reduction was IFNγ- and CD1d-dependent, similar to CT as adjuvant [165]. In conclusion, α-GalCer is an effective mucosal adjuvant for oral immunization and can enhance the mucosal responses of IgA and Th1 in mice, but its safety and efficacy in humans still warrant further evaluation. In addition to its impressive oral adjuvant effects in mice, α-GalCer has been tested in clinical trials for the treatment of cancer and hepatitis, in which its safety has been assessed [14].
3.5 Synthetic Toll-Like Receptor (TLR)-Agonist Molecules
As mentioned earlier, TLR molecules have been the target of many new mucosal vaccine candidates. Targeting one or more TLR(s) might activate sensors of innate TLR pathogens and promote intracellular signaling cascades that lead to upregulation of the production of chemokines and cytokines required for DC maturation, which results in increased magnitude and quality of immune responses [93, 166]. Some synthetic TLR ligands could also activate TLR signals and subsequently promote immune responses, which have been exploited as potential adjuvants of mucosal vaccines. For example, incorporation of the TLR4 agonist monophosphoryl lipid A (MPL) into nanoparticle vaccines could contribute to triggering TLR signaling with mucosal DCs and subsequently improve the capture efficiency of vaccines [160]. The TLR 7/8 agonists R848 have showed great potential as oral vaccine adjuvants because they can directly activate APCs and enhance both humoral and cellular immune responses, especially Th1 responses [167]. According to Borducchi et al., oral administration of Ad26/MVA combined with the TLR7 agonist GS-986 could decrease the level of SIV viral DNA in lymph nodes and peripheral blood, as well as control and delay virologic rebound following antiretroviral therapy discontinuation in SIV-infected Rhesus Monkeys [168].
In addition, CpG oligodeoxynucleotides (CpG-ODN) are another promising synthetic TLR-agonist adjuvant. They are short single-stranded synthetic DNA molecules that can activate the immune system and have been found to be effective in the prevention and treatment of infectious diseases, allergies, and cancers [16, 169]. CpG-ODN, a ligand of TLR9, can activate TLR9 on B-lymphocytes and DCs, showing potent activity in stimulating antigen presentation and inducing antigen-specific immune response towards the Th1 phenotype [170]. Alignani et al. reported CpG-ODN-loaded ovalbumin (OVA) induced specific mucosal and systemic immune response in mice after oral administration [171]. CpG-ODN has different classes, such as CpG-ODN 2007 [172], CpG-ODN 1668 [173], and CpG-ODN 1826 [174], and has also shown potent mucosal adjuvant activity. Hjelm et al. [175] used a panel of TLR agonists (PIC [TLR3], FLAG [TLR5], GARD [TLR7], CpG [TLR9], CpG-ISS [CpG 1018, alternate CpG motif, TLR9], and CL097 [TLR7/8]) as adjuvants combined with Norwalk VLPs (NV VLPs) coadministered to mice through intranasal and oral routes to determine the mucosal adjuvant activity of these immunomodulators. Of these, intranasal co-delivery of VLPs with TLR7 or TLR9 agonists (i.e., GARD or CpG) produced the most robust and broad-spectrum immune response, but oral administration with other TLR agonists (i.e., PIC, FLAG, and CL097) could not consistently enhance VLP-specific immune responses in mice.
According to our knowledge, there are no human trials using TLR agonists as oral vaccine adjuvants. These above studies are preclinical studies, indicating that TLR plays an important role in inducing immune response in the oral route.
3.6 Composite Non-biogenic Material Adjuvants
Through chemical modification or nanotechnology, the physicochemical properties of non-biogenic adjuvant materials (or polymeric nanoparticles) can be improved by combining them with another non-biogenic adjuvant material. Their advantages can be complementary, which is beneficial to enhance the interaction between nanoparticle adjuvant and intestinal endocytosis pathways [176, 177]. For instance, UEA-1 is the M-cell selective molecular signature, which could exclusively adhere to M cells, and MPL is a TLR agonist. With a combination of UEA-1 and MPL, Ma et al. [160] reported that the composite material, UEA-MPL-conjugated PLGA-lipid nanoparticles, can be effectively transported by M cells and captured by mucosal DCs, showing the potential of an attractive oral vaccine delivery system for boosting oral immunity. Ma et al. found that OVA-UEA-MPL/lipid nanoparticles stimulated the most effective mucosal IgA and serum IgG antibodies during oral vaccination [160]. Sarti et al. used MPL-conjugated PLGA nanoparticles as an oral adjuvant of OVA in mice [178], and compared with the control formulation group, it generated significantly higher IgA titers, which indicated that MPL-PLGA nanoparticles had the ability to induce mucosal immunity. Salman et al. reported that mannosamine-coated poly(anhydride) nanoparticles as an oral composite adjuvant of OVA induced strong, long-lasting systemic and mucosal immune responses than the non-conjugated vectors [179]. Moreover, Mishra et al. demonstrated that LTA (Lotus tetragonolobus from Winged or Asparagus pea)-anchored PLGA nanoparticles could elicit strong mucosal and systemic response and hence could be a promising M cell-targeting adjuvant for oral mucosal immunization against hepatitis B [180]. Alginate-coated chitosan nanoparticles are of interest because of their great stability and immunostimulatory properties. They can effectively transport antigens into the M cells and subsequently induce significant immune responses in serum IgG and mucosal sIgA levels [156, 181]. Borges et al. demonstrated that alginate-coated chitosan nanoparticles showed potential as a delivery system for oral recombinant HBsAg [182]. Most recently, Yu et al. demonstrated that alginate-chitosan coated layered double hydroxide nanocomposites (ALG-CHT-LDHs) showed great potential in oral protein vaccine delivery [157]. In addition, Taha-Abdelaziz et al. reported that oral administration of PLGA-encapsulated CpG ODN, and Campylobacter jejuni lysate reduced cecal colonization by C. jejuni in chickens [183].
The above studies show that the physicochemical properties of single non-biogenic adjuvant material can be improved by comprehensive combination of double or triple, or even quadruple, adjuvant materials through nanotechnology, so as to correctly match the size, electric charge, hydrophobicity, and other physicochemical properties of antigen, and so that antigen can cross the mucosal barriers and target APCs [184]. Through the appropriate combination of a variety of non-biogenic adjuvant materials, its advantages could be developed and its disadvantages could be avoided, so as to construct a universal and powerful oral vaccine carrier or adjuvant.
4 Biogenic and Non-biogenic Combined Composite Material for Oral Adjuvants
Nowadays, using conjugation techniques to combine biogenic adjuvants (such as M cell-targeting peptides, bacterial flagellin, or cytokines, etc.) with other non-biogenic adjuvants to create composite adjuvants can give full play to the advantages of the activity of each adjuvant, thus enhancing the overall activity of adjuvants (Table 3). The composite adjuvant, PLGA microparticles coated with chitosan-coupled M cell-homing peptide (CKS9), can strengthen the targeting ability to M cells, and the mucosal and systemic immune responses were induced when it was used to deliver swine dysentery vaccine [185]. As mentioned previously, PNPs are natural mucoadhesive polymers that could efficiently deliver antigens to the GALT [179], and flagellar protein possesses potent immune adjuvant activity. Renu et al. designed a Salmonella subunit vaccine (OMPs-F-PNPs) that consisted of PNPs containing immunogenic Salmonella OMPs and entrapped flagellar (F) protein and surface F-protein-coated PNPs. The vaccine could induce specific immune response to mitigate Salmonella colonization in the intestines of chickens vaccinated orally [125]. Yang et al. used PLGA nanoparticles combined with cytokine as adjuvant to deliver DNA vaccine of foot and mouth disease, which significantly enhanced its immunogenicity than naked DNA [186]. Generally, the conjugation of biogenic adjuvant and non-biogenic adjuvant as composite adjuvants is another frequently used and promising way to assist the delivery of oral vaccine and enhance its immunogenicity.
5 Concluding Remarks, Challenges, and Future Perspectives
The complex and harsh environment in GIT leads to the weak immunogenicity of peroral mucosal vaccines. Preparation of effective adjuvants to enhance the immune response is an integral part of the development of oral vaccines. Over the past few decades, several biogenic and/or non-biogenic adjuvants have been used in the trials of various peroral mucosal vaccines to enhance their immune responses. At present, among the aforementioned adjuvant candidates of peroral vaccines, only dmLT has undergone human clinical trials and further passed clinical phase I and II trials [187, 188], while other adjuvant candidates are still in animal (or veterinary or aquatic) experimental stage (Fig. 2). In this review, some adjuvants have been tested on farm animals, such as pigs, birds, fish, etc., to develop veterinary vaccines (or adjuvants) (Fig. 2). For the development of veterinary vaccines (or adjuvants), the target animals are the most ideal animal models. Mice (especially BALB/c mice) are commonly used animal models for preclinical trials of adjuvants (or vaccines) in animals or humans (Fig. 2). Animal models play a critical role in the in vivo study of the immunology and pharmacology of oral adjuvant (or vaccines) candidates, as well as the evaluation of potential applications in humans. However, it is undeniable that many animal models used in oral adjuvant (or vaccines) tests have limited predictive value for the human response to oral adjuvants (or vaccines) in terms of both efficacy and toxicology [189]. Among the examples of oral adjuvants (or candidates) we summarized (Tables 1, 2, 3), there were few reports focusing on the purposeful selection of animal models to evaluate the efficacy of oral adjuvants. Many researchers chose animal models for their animal experiments based on the disease types they were studying, instead of the oral adjuvants, which made it difficult to translate the positive effects of oral adjuvants on animal models to humans and required considerable analysis and debate. It is necessary to improve the existing animal models to make them more predictive for humans.
List of oral adjuvant candidates developed and their corresponding in vivo tests. According to the physicochemical properties, oral adjuvants could be divided into biogenic, non-biogenic, and a biogenic and non-biogenic combined composite. In vivo tests for oral adjuvant development have involved humans, rabbits, fish, rodents, pigs, primates, canines, and chickens. No connection means the in vivo test has not yet been carried out. α-GalCer alpha-Galactosylceramide, c-di-AMP 3′5′-cyclic di-adenosine monophosphate, CKS9 M cell-targeting peptide, Co1 M cell-specific peptide ligands, CpG-ODN CpG oligodeoxynucleotides, DCpep dendritic cell-targeting peptide, dmLT double-mutant heat-labile toxin, FnBPA fibronectin binding protein A, GM-CSF granulocyte-macrophage colony-stimulating factor, MCS NPs mannosylated chitosan nanoparticles, MDP muramyl dipeptide, mmCT multiple mutant cholera toxin, MPL monophosphoryl lipid A, O-2′-HACC O-2′-hydroxypropyltrimethyl ammonium chloride chitosan, PLG poly(d,l-lactide-co-glycolide), PLGA poly(d,l-lactic-co-glycolic acid), RANKL receptor activator of NF-kB ligand, RCK Salmonella resistance to complement killing, SMIPs small molecular immunomodulatory proteins, TMC trimethyl chitosan, UEA-1 ulex europaeus agglutinin-1
An obvious advantage of biogenic adjuvants is that they can be constructed or optimized by genetic engineering. On the one hand, gene editing technology is used to remove their toxicity and optimize their adjuvant performance, such as from LT to dmLT (R192G/L211A) and from CT to mmCT. On the other hand, the biogenic adjuvants could be fused with the target antigens by DNA recombination technology to construct the fusion protein vaccines. In addition, most biogenic adjuvants could be encoded and expressed in beneficial bacterial strains, such as probiotics (e.g., LAB), attenuated live bacteria, or gut commensal bacteria, as a protective strategy across the GIT against degradation from gastric acid and proteases, etc. This is also another promising way to develop oral vaccines. Ideally, adjuvants should not induce adaptive immune responses against themselves, but should promote appropriate immune response to accompanying antigens [190, 191]. However, some well-known protein-based oral adjuvants listed above, including FliC [192, 193], CTB [194], FnBPA [51], PorA [195], and even the well-studied dmLT [196], have been reported to produce a certain degree of immune response against themselves in the host, thereby potentially affecting their effectiveness as adjuvants. However, FljB has been reported to produce no immune response against itself [40], and an appropriate oral dose of dmLT is still safe, well tolerated, and reasonably immunogenic [196].
Using non-biogenic adjuvants, protein antigens could be coated in a variety of ways, such as lipidation, nanoparticle encapsulation (using polymersomes, for example), adsorption and conjugation to polymer-based microparticles/nanoparticles (using PLGA, chitosan, alginate, etc.) and/or additive/synergistic admixture [167]. In addition, combining two or more types of adjuvant materials (including biogenic and non-biogenic materials) to construct composite adjuvants can make up for inherent flaws of some biogenic adjuvants, such as their short half-life and ease of degradation in GIT, and strengthen their immune effects in the intestinal tract. There are also some nanomaterials that can control the slow release of vaccines. For example, some TLR ligands are often chimeric with other nano-adjuvant materials, therefore the adjuvant effect is better.
Undoubtedly, in order to exert the adjuvant activity for peroral mucosal vaccines, more attention should be focused on the endogenous immune activation mechanisms of adjuvants and the immunological and pharmacological relationship among adjuvants, vaccines (or antigens) and the host gastrointestinal mucosal immune system. Although there are various adjuvant strategies, they should all be studied in detail before selecting the optimum formulation. The formulation of vaccines and adjuvants should not only maintain the immunogenicity of the vaccine but also protect their adjuvant activity. This is an issue that needs meticulous consideration when designing oral vaccines.
The development of oral adjuvants still presents many challenges. As mentioned earlier, the GIT is a complex and harsh environment, which leads to the instability of adjuvants, especially biogenic adjuvants, and hinders the interaction between oral adjuvants and intestinal epithelial cells. On the other hand, the low proportion of M cells in the intestinal epithelium would limit the effect of M cell-mediated adjuvants. However, we believe that more and more oral adjuvants, similar to RANKL, that can induce M-cell differentiation will emerge in the future to change this dilemma. The potential safety concerns of adjuvants, such as cytokine-derived adjuvants [93], are another challenge. The effective targeting, pharmacokinetics and nanotoxicology of some potential oral adjuvants need to be further evaluated.
As mentioned previously, the targeting peptides that target intestinal immune cells (or receptor proteins on their cell surface) can improve the binding ability of antigens to bind to intestinal DCs or M cells (or receptors). Therefore, in addition to the targeting peptides of human and/or mouse intestinal immune cells (and their receptor proteins), some researchers are trying to screen and identify specific M cells or DC-binding peptides of other animal species for studies in veterinary or comparative medicine by using the cell-based phage display technique combined with high-throughput sequencing; for instance, the chicken DC-binding peptide (SPHLHTSSPWER, named SP) [197] and the porcine TLR2-targeting peptide ligand (NAGHLSQ) [198] [porcine TLR2 is highly expressed in M cells and plays an important role in pig mucosal immune responses]. Aiming at M cells or DCs (or receptor proteins on their cell surface), it will be a trend to develop more targeting peptides for different animal species, especially in the prevention and control of veterinary infectious diseases. Furthermore, with further understanding of the mechanisms of action of some less-studied candidate adjuvants, such as muramyl dipeptide and tuftsin fusion protein (MT) [54, 55], N. meningitidis PorA [57], c-di-AMP [58], RCK protein [62], etc., these may be the future development direction of oral adjuvants.
With the development of oral adjuvants in recent years, it is believed that more reasonable and effective oral adjuvants will appear in the future and hence solve the challenges mentioned.
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The authors acknowledge the assistance of Xiaojie Chen, Leilei Liu and Tingting Shen from Zhaoqing University in the preparation and revision of this article.
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Financial support for this study was provided by the National Natural Science Foundation of China (Grant no. 32102703), Basic and Applied Basic Research Joint Fund of Guangdong Province (2019A1515111186), National College Students’ Innovation and Entrepreneurship Training Plan Program in 2021 (202110580008), College Students’ Innovation and Entrepreneurship Training Plan Program of Guangdong Province (S202010580051, S202210580052), Fund of Construction of Ecological Poultry Industry Technology System in Guizhou Province (Functional Laboratory of Disease Prevention and Control), and the Scientific Research Start-up Fund of Zhaoqing University (611/180160), Social Public Welfare Science and Technology Research Project of Zhongshan City (2019B2022).
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Bingming Ou, Ying Yang, Haihui Lv, Xin Lin, and Minyu Zhang declare that they have no competing interests.
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Ou, B., Yang, Y., Lv, H. et al. Current Progress and Challenges in the Study of Adjuvants for Oral Vaccines. BioDrugs 37, 143–180 (2023). https://doi.org/10.1007/s40259-022-00575-1
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DOI: https://doi.org/10.1007/s40259-022-00575-1